Publications

Huang, X., et al. (2017). "Modularized Gold Nanocarriers for TAT-Mediated Delivery of siRNA." Small 13(8):1602473.

                Targeted delivery of siRNA controlled by near-infrared light using hollow gold nanoshells has been demonstrated in cancer and stem cells models. Here, a universal surface module and several functionalization rules for the maximized delivery of short nucleic acids (here, siRNA) applicable for diverse gold nanocarriers are described. Streptavidin is devised as a handle to assemble biotinylated cell penetrating peptides (e.g., transactivating transcriptional activator (TAT)), as well as an insulator between the positive charge of TAT and the dense negative charge of RNA. However, direct linking of streptavidin to functional siRNA inhibits its silencing activity. The approach then involves the orthogonal assembly of two types of RNA strands: one with biotin modification for cell targeting and penetration (scaffold RNA); the other without biotin as functional RNA (i.e., siRNA). Initially, flexible single-stranded RNA is used for dense surface-packing, followed by hybridization with the complementary RNA strand to maximize the assembly of the targeting peptide for cellular uptake and siRNA delivery. This orthogonal approach for the delivery of short oligonucleotides, together with novel surface functionalization rules discovered here, should enable the use of these materials for nanomedicinal research and applications.

 

Morales DP., et al. (2017).  "Affinity-Based Assembly of Peptides on Plasmonic Nanoparticles Delivered Intracellularly with Light Activated Control." Bioconjugate Chemistry 28(7): 1816–1820.

                We report a universal strategy for functionalizing near-infrared light-responsive nanocarriers with both a peptide “cargo” and an orthogonal cell-penetrating peptide. Modularity of both the cargo and the internalization peptide attachment is an important feature of these materials relying on the robust affinity of polyhistidine tags to nitrilotriacetic acid in the presence of nickel as well as the affinity of biotin labeled peptides to streptavidin. Attachment to the gold surface uses thiol-labeled scaffolds terminated with the affinity partner. These materials allow for unprecedented spatiotemporal control over the release of the toxic α-helical amphipathic peptide (KLAKLAK)2 which disrupts mitochondrial membranes and initiates apoptotic cell death. Laser treatment at benign near-infrared wavelengths releases peptide from the gold surface as well as breaches the endosome barrier for cytosolic activity (with 105-fold improved response to peptide activity over the free peptide) and can be monitored in real time.

 

Woodcock, C., et al. (2017).  "Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition." Biochemistry

                Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5′-GANTC-3′ in single-stranded and hemimethylated double-stranded DNA. We report the  Km,  kcat,  kmethylation, and  Kd for single-stranded and hemimethylated substrates, revealing discrimination of 107-fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

 

Woodcock, C., et al. (2016).  "Rational Manipulation of DNA Methylation by Using Isotopically Reinforced Cytosine." ChemBioChem 17: 2018–2021.

                The human DNA methyltransferase 3A (DNMT 3A) is responsible for de novo epigenetic regulation, which is essential for mammalian viability and implicated in diverse diseases. All DNA cytosine C5 methyltransferases follow a broadly conserved catalytic mechanism. We investigated whether C5 belimination contributes to the rate-limiting step in catalysis by DNMT3A and the bacterial M.HhaI by using deuterium substitutions of C5 and C6 hydrogens. This substitution caused a 1.59–1.83 fold change in the rate of catalysis, thus suggesting that b-elimination is partly rate-limiting for both enzymes. We used a multisite substrate to explore the consequences of slowing b-elimination during multiple cycles of catalysis. Processive catalysis was slower for both enzymes, and deuterium substitution resulted in DNMT 3A dissociating from its substrate. The decrease in DNA methylation rate by DNMT 3A provides the basis of our ongoing efforts to alter cellular DNA methylation levels without the toxicity of currently used methods.

 

Huang, X., et al. (2016). "Light-Patterned RNA Interference of 3D-Cultured Human Embryonic Stem Cells." Adv Mater 28(48):10732–10737.

                A new method of spatially controlled gene regulation in 3D-cultured human embryonic stem cells is developed using hollow gold nanoshells (HGNs) and near-infrared (NIR) light. Targeted cell(s) are discriminated from neighboring cell(s) by focusing NIR light emitted from a two-photon microscope. Irradiation of cells that have internalized HGNs releases surface attached siRNAs and leads to concomitant gene downregulation.

 

Huang, X., et al. (2015). "Light-activated RNA interference in human embryonic stem cells." Biomaterials 63: 70–79.

                We describe a near infrared (NIR) light-activated gene silencing method in undifferentiated human embryonic stem cell (hESC) using a plasmonic hollow gold nanoshell (HGN) as the siRNA carrier. Our modular biotin-streptavidin coupling strategy enables positively charged TAT-peptide to coat oligonucleotides-saturated nanoparticles as a stable colloid formation. TAT-peptide coated nanoparticles with dense siRNA loading show efficient penetration into a wide variety of hESC cell lines. The siRNA is freed from the nanoparticles and delivered to the cytosol by femtosecond pulses of NIR light with potentially exquisite spatial and temporal control. The effectiveness of this approach is shown by targeting GFP and Oct4 genes in undifferentiated hESC (H9). The accelerated expression of differentiation markers for all three germ layers resulting from Oct4 knockdown confirms that this method has no detectable adverse effects that limit the range of differentiation. This biocompatible and NIR laser activated patterning method makes possible single cell resolution of siRNA delivery for diverse studies in stem cell biology, tissue engineering and regenerative medicine.

 

Morales DP., et al. (2015).  "Near-IR mediated intracellular uncaging of NO from cell targeted hollow gold nanoparticles." ChemComm 51(100):17692–17695.

                We demonstrate modulation of nitric oxide release in solution and in human prostate cancer cells from a thiol functionalized cupferron (TCF) absorbed on hollow gold nanoshells (HGNs) using near-infrared (NIR) light. NO release from the TCF–HGN conjugates occurs through localized surface heating due to NIR excitation of the surface plasmon. Specific HGN targeting is achieved through cell surface directed peptides, and excitation with tissue penetrating NIR light provides unprecedented spatio-temporal control of NO delivery to biological targets.

 

Morales DP., et al. (2014).  "Targeted Intracellular Delivery of Proteins with Spatial and Temporal Control." Molecular Pharmaceutics 12(2): 600–609.

                While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.

 

Pollak, A. J., et al. (2014). "Distinct facilitated diffusion mechanisms by E. coli Type II restriction endonucleases." Biochemistry53(45): 7028–7037.

                The passive search by proteins for particular DNA sequences involving non-specific DNA is essential for gene regulation, DNA repair, phage defense, and diverse epigenetic processes. Distinct mechanisms contribute to these searches and it remains unresolved which mechanism or blend of mechanisms best suits a particular protein and more importantly, its biological role. To address this we compare the translocation properties of two well-studied bacterial restriction endonucleases (ENases), EcoRI and EcoRV. These dimeric, magnesium-dependent enzymes hydrolyze related sites (EcoRI ENase: 5'-GAATTC-3' and EcoRV ENase: 5'-GATATC-3') leaving overhangs and blunt DNA segments respectively. Here we demonstrate that the extensive sliding by EcoRI ENase, involving sliding up to ~600 bp prior to dissociating from the DNA, contrasts with a larger reliance on hopping mechanism(s) by EcoRV ENase. The mechanism displayed by EcoRI ENase results in a highly thorough search of DNA whereas the EcoRV ENase mechanism results in an extended, yet less rigorous interrogation of DNA sequence space. We describe how these mechanistic distinctions are complemented by other aspects of these endonucleases, such as the ten-fold higher in vivo concentrations of EcoRI ENase compared to EcoRV ENase. Further, we hypothesize that the highly diverse enzyme arsenal which bacteria employ against foreign DNA involves seemingly similar enzymes which rely on distinct but complementary search mechanisms. Our comparative approach reveals how different proteins utilize distinct site locating strategies.

 

Pollak, A. J., et al. (2014). "DNA looping provides for "intersegmental hopping" by proteins: a mechanism for long-range site localization." J Mol Biol 426(21): 3539-3552.

                Studies on how transcription factors and DNA modifying enzymes passively locate specific sites on DNA have yet to be reconciled with a sufficient set of mechanisms that can adequately account for the efficiency and speed of this process. This is especially true when considering that these DNA binding/modifying proteins have diverse levels of both cellular copy numbers and genomic recognition site densities. The monomeric bacterial DNA adenine methyltransferase (Dam) is responsible for the rapid methylation of the entire chromosome (with only ~100 Dam copies per cell) and the regulated methylation of closely spaced sites that controls the expression of virulence genes in several human pathogens. Provocatively, we find that Dam travels between its recognition sites most efficiently when those sites are ~500bp apart. We propose that this is manifested by Dam moving between distal regions on the same DNA molecule, which is mediated by DNA looping, a phenomenon we designate as intersegmental hopping. Importantly, an intermediate found in other systems including two simultaneously bound, looped DNA strands is not involved here. Our results suggest that intersegmental hopping contributes to enzymatic processivity (multiple modifications), which invoke recent reports demonstrating that DNA looping can assist in site finding. Intersegmental hopping is possibly used by other sequence-specific DNA binding proteins, such as transcription factors and regulatory proteins, given certain biological context. While a general form of this mechanism is proposed by many research groups, our consideration of DNA looping in the context of processive catalysis provides new mechanistic insights and distinctions.

 

Huang, X., et al. (2014). "Modular plasmonic nanocarriers for efficient and targeted delivery of cancer-therapeutic siRNA." Nano Lett 14(4): 2046-2051.

                We have combined a versatile and powerful route to deliver nucleic acids with peptide-based cell-specific targeting. siRNA targeting the polo-like kinase gene is in clinical trials for cancer treatment, and here we deliver this RNA selectively to cancer cells displaying the neuropilin-1 epitope using gold nanoshells. Release of the siRNA from the nanoparticles results from irradiation with a pulsed near-infrared laser, which also provides efficient endosomal escape within the cell. As a result, our approach requires 10-fold less material than standard nucleic acid transduction materials and is significantly more efficient than other particle-based methods. We also describe a particle-nucleic acid design that does not rely on modified RNA, thereby making the preparation of these materials more efficient and much less expensive. These improvements, when combined with control over when and where the siRNA is released, could provide the basis for diverse cell biological studies.

 

Matje, D. M., et al. (2013). "Distal structural elements coordinate a conserved base flipping network." Biochemistry 52(10): 1669-1676.

                One of the most dramatic illustrations of enzymatic promotion of a high-energy intermediate is observed in DNA modification and repair enzymes where an individual base is rotated (flipped) 180 degrees around the deoxyribose-phosphate backbone and into the active site. While the end states have been extensively characterized, experimental techniques have yet to yield a full description of the base flipping process and the role played by the enzyme. The C5 cytosine methyltransferase M.HhaI coordinates an ensemble of reciprocal DNA and enzyme rearrangements to efficiently flip the target cytosine from the DNA helix. We sought to understand the role of individual amino acids during base flipping. Our results demonstrate that M.HhaI initiates base flipping before closure of the catalytic loop and utilizes the conserved serine 85 in the catalytic loop to accelerate flipping and maintain distortion of the DNA backbone. Serine 87, which forms specific contacts within the DNA helix after base flipping, is not involved in the flipping process or in maintaining the catalytically competent complex. At the base of the catalytic loop, glycine 98 acts as a hinge to allow conformational dynamism of the loop and mutation to alanine inhibits stabilization of the closed loop. Our results illustrate how an enzyme utilizes numerous, distal residues in concert to transform substrate recognition into catalysis.

 

Matje, D. M., et al. (2013). "Enzyme-promoted base flipping controls DNA methylation fidelity." Biochemistry 52(10): 1677-1685.

                A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme-substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.HhaI has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein-DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.

 

Bonham, A. J., et al. (2013). "STAT1:DNA sequence-dependent binding modulation by phosphorylation, protein:protein interactions and small-molecule inhibition." Nucleic Acids Research 41(2): 754-763.

                The DNA-binding specificity and affinity of the dimeric human transcription factor (TF) STAT1, were assessed by total internal reflectance fluorescence protein-binding microarrays (TIRF-PBM) to evaluate the effects of protein phosphorylation, higher-order polymerization and small-molecule inhibition. Active, phosphorylated STAT1 showed binding preferences consistent with prior characterization, whereas unphosphorylated STAT1 showed a weak-binding preference for one-half of the GAS consensus site, consistent with recent models of STAT1 structure and function in response to phosphorylation. This altered-binding preference was further tested by use of the inhibitor LLL3, which we show to disrupt STAT1 binding in a sequence-dependent fashion. To determine if this sequence-dependence is specific to STAT1 and not a general feature of human TF biology, the TF Myc/Max was analysed and tested with the inhibitor Mycro3. Myc/Max inhibition by Mycro3 is sequence independent, suggesting that the sequence-dependent inhibition of STAT1 may be specific to this system and a useful target for future inhibitor design.

 

Matje, D. M., et al. (2013). "Distal Structural Elements Coordinate a Conserved Base Flipping Network." Biochemistry 52(10): 1669-1676.

                One of the most dramatic illustrations of enzymatic promotion of a high-energy intermediate is observed in DNA modification and repair enzymes where an individual base is rotated (flipped) 180 degrees around the deoxyribose phosphate backbone and into the active site. While the end states have been extensively characterized, experimental techniques have yet to yield a full description of the base flipping process and the role played by the enzyme. The CS cytosine methyltransferase M.HhaI coordinates an ensemble of reciprocal DNA and enzyme rearrangements to efficiently flip the target cytosine from the DNA helix. We sought to understand the role of individual amino acids during base flipping. Our results demonstrate that M.HhaI initiates base flipping before closure of the catalytic loop and utilizes the conserved serine 85 in the catalytic loop to accelerate flipping and maintain distortion of the DNA backbone. Serine 87, which forms specific contacts within the DNA helix after base flipping, is not involved in the flipping process or in maintaining the catalytically competent complex. At the base of the catalytic loop, glycine 98 acts as a hinge to allow conformational dynamism of the loop and mutation to alanine inhibits stabilization of the closed loop. Our results illustrate how an enzyme utilizes numerous, distal residues in concert to transform substrate recognition into catalysis.

 

Matje, D. M., et al. (2013). "Enzyme-Promoted Base Flipping Controls DNA Methylation Fidelity." Biochemistry 52(10): 1677-1685.

                A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.Hhal has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein-DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.

 

Cohn, E. P. M. T., et al. (2012). "A New Strategy for Detection and Development of Tractable Telomerase Inhibitors." Journal of Medicinal Chemistry 55(8): 3678-3686.

                Despite intense academic and industrial efforts and innumerable in vitro and cell studies, no small-molecule telomerase inhibitors have emerged as drugs. Insufficient understanding of enzyme structure and mechanisms of interdiction coupled with the substantial complexities presented by its dimeric composition have stalled all progress toward small-molecule therapeutics. Here we challenge the assumption that human telomerase provides the best platform for inhibitor development by probing a monomeric Tetrahymena telomerase with six tool compounds. We find BIBR-1532 (2) and MST-312 (5) inhibit only human telomerase, whereas beta-R (1), THyF (3), TMPyP4 (6), and EGCG (4) inhibit both enzymes. Our study demonstrates that some small-molecule scaffolds can be easily surveyed with in vitro studies using Tetrahymena telomerase, a finding that could lead to more tractable inhibitors with a greater potential for development given the more precise insights that can be gleaned from this more easily expressed and assayed monomeric enzyme.

 

Holz-Schietinger, C. and N. O. Reich (2012). "RNA modulation of the human DNA methyltransferase 3A." Nucleic Acids Research 40(17): 8550-8557.

                DNA methyltransferase 3A (DNMT3A) is one of two human de novo DNA methyltransferases essential for transcription regulation during cellular development and differentiation. There is increasing evidence that RNA plays a role in directing DNA methylation to specific genomic locations within mammalian cells. Here, we describe two modes of RNA regulation of DNMT3A in vitro. We show a single-stranded RNA molecule that is antisense to the E-cadherin promoter binds tightly to the catalytic domain in a structurally dependent fashion causing potent inhibition of DNMT3A activity. Two other RNA molecules bind DNMT3A at an allosteric site outside the catalytic domain, causing no change in catalysis. Our observation of the potent and specific in vitro modulation of DNMT3A activity by RNA supports in vivo data that RNA interacts with DNMT3A to regulate transcription.

 

Holz-Schietinger, C., et al. (2012). "Mutations in DNA Methyltransferase (DNMT3A) Observed in Acute Myeloid Leukemia Patients Disrupt Processive Methylation." Journal of Biological Chemistry 287(37): 30941-30951.

                DNA methylation is a key regulator of gene expression and changes in DNA methylation occur early in tumorigenesis. Mutations in the de novo DNA methyltransferase gene, DNMT3A, frequently occur in adult acute myeloid leukemia patients with poor prognoses. Most of the mutations occur within the dimer or tetramer interface, including Arg-882. We have identified that the most prevalent mutation, R882H, and three additional mutants along the tetramer interface disrupt tetramerization. The processive methylation of multiple CpG sites is disrupted when tetramerization is eliminated. Our results provide a possible mechanism that accounts for how DNMT3A mutations may contribute to oncogenesis and its progression.

 

Laurence, T. A., et al. (2012). "Robust SERS Enhancement Factor Statistics Using Rotational Correlation Spectroscopy." Nano Lett 12(6): 2912-2917.

                We characterize the distribution of surface-enhanced Raman spectroscopy (SERS) enhancement factors observed in individual hot spots of single Ag "nanocapsules", encapsulated Ag nanoparticle dimers formed via controlled nanoparticle linking, polymer encapsulation, and small molecule infusion. The enhancement factors are calculated for over 1000 individual nanocapsules by comparing Raman scattering intensities of 4-mercaptobenzoic acid (MBA) measured from single SERS hot spots to intensities measured from high-concentration solutions of MBA. Correlation spectroscopy measurements of the rotational diffusion identify nanocapsules with signals dominated by single hot spots via their strong polarization response. Averaging over the entire surface of the nanocapsules, the distribution of enhancement factors is found to range from 10(6) to 10(8), with a mean of 6 X 10(6). Averaging only over nanoparticle junctions (where most SERS signals are expected) increases this average value to 10(8), with a range from 2 X 10(7) to 2 X 10(9). This significant statistical sampling shows that very high SERS enhancement factors can be obtained on a consistent basis using nanoparticle linking.

 

Matje, D. M. and N. O. Reich (2012). "Molecular Drivers of Base Flipping During Sequence-Specific DNA Methylation." Chembiochem 13(11): 1574-1577.

               

Pollak, A. J. and N. O. Reich (2012). "Proximal Recognition Sites Facilitate Intrasite Hopping by DNA Adenine Methyltransferase Mechanistic Exploration of Epigenetic Gene Regulation." Journal of Biological Chemistry 287(27): 22873-22881.

                The methylation of adenine in palindromic 5'-GATC-3' sites by Escherichia coli Dam supports diverse roles, including the essential regulation of virulence genes in several human pathogens. As a result of a unique hopping mechanism, Dam methylates both strands of the same site prior to fully dissociating from the DNA, a process referred to as intrasite processivity. The application of a DpnI restriction endonuclease-based assay allowed the direct interrogation of this mechanism with a variety of DNA substrates. Intrasite processivity is disrupted when the DNA flanking a single GATC site is longer than 400 bp on either side. Interestingly, the introduction of a second GATC site within this flanking DNA reinstates intrasite methylation of both sites. Our results show that intrasite methylation occurs only when GATC sites are clustered, as is found in gene segments both known and postulated to undergo in vivo epigenetic regulation by Dam methylation. We propose a model for intrasite methylation in which Dam bound to flanking DNA is an obligate intermediate. Our results provide insights into how intrasite processivity, which appears to be context-dependent, may contribute to the diverse biological roles that are carried out by Dam.

 

Holz-Schietinger, C., et al. (2011). "Oligomerization of DNMT3A Controls the Mechanism of de Novo DNA Methylation." Journal of Biological Chemistry 286(48): 41479-41488.

                DNMT3A is one of two human de novo DNA methyltransferases essential for regulating gene expression through cellular development and differentiation. Here we describe the consequences of single amino acid mutations, including those implicated in the development of acute myeloid leukemia (AML) and myelodysplastic syndromes, at the DNMT3A.DNMT3A homotetramer and DNMT3A.DNMT3L heterotetramer interfaces. A model for the DNMT3A homotetramer was developed via computational interface scanning and tested using light scattering and electrophoretic mobility shift assays. Distinct oligomeric states were functionally characterized using fluorescence anisotropy and steady-state kinetics. Replacement of residues that result in DNMT3A dimers, including those identified in AML patients, show minor changes in methylation activity but lose the capacity for processive catalysis on multisite DNA substrates, unlike the highly processive wild-type enzyme. Our results are consistent with the bimodal distribution of DNA methylation in vivo and the loss of clustered methylation in AML patients. Tetramerization with the known interacting partner DNMT3L, rescues processive catalysis, demonstrating that protein binding at the DNMT3A tetramer interface can modulate methylation patterning. Our results provide a structural mechanism for the regulation of DNMT3A activity and epigenetic imprinting.

 

Matje, D. M., et al. (2011). "Determinants of Precatalytic Conformational Transitions in the DNA Cytosine Methyltransferase M.HhaI." Biochemistry 50(9): 1465-1473.

                The DNA methyltransferase M.HhaI is an excellent model for understanding how recognition of a nucleic acid substrate is translated into site-specific modification. In this study, we utilize direct, real-time monitoring of the catalytic loop position via engineered tryptophan fluorescence reporters to dissect the conformational transitions that occur in both enzyme and DNA substrate prior to methylation of the target cytosine. Using nucleobase analogues in place of the target and orphan bases, the kinetics of the base flipping and catalytic loop closure rates were determined, revealing that base flipping precedes loop closure as the rate-determining step prior to methyl transfer. To determine the mechanism by which individual specific hydrogen bond contacts at the enzyme DNA interface mediate these conformational transitions, nucleobase analogues lacking hydrogen bonding groups were incorporated into the recognition sequence to disrupt the major groove recognition elements. The consequences of binding, loop closure, and catalysis were determined for four contacts, revealing large differences in the contribution of individual hydrogen bonds to DNA recognition and conformational transitions on the path to catalysis. Our results describe how M.HhaI utilizes direct readout contacts to accelerate extrication of the target base that offer new insights into the evolutionary history of this important class of enzymes.

 

Menezes, S., et al. (2011). "Formation of m(2)G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14." Nucleic Acids Research 39(17): 7641-7655.

                The modified nucleosides N-2-methylguanosine and N-2(2)-dimethylguanosine in transfer RNA occur at five positions in the D and anticodon arms, and at positions G6 and G7 in the acceptor stem. Trm1 and Trm11 enzymes are known to be responsible for several of the D/anticodon arm modifications, but methylases catalyzing post-transcriptional m(2)G synthesis in the acceptor stem are uncharacterized. Here, we report that the MJ0438 gene from Methanocaldococcus jannaschii encodes a novel S-adenosylmethionine-dependent methyltransferase, now identified as Trm14, which generates m(2)G at position 6 in tRNA(Cys). The 381 amino acid Trm14 protein possesses a canonical RNA recognition THUMP domain at the amino terminus, followed by a gamma-class Rossmann fold amino-methyltransferase catalytic domain featuring the signature NPPY active site motif. Trm14 is associated with cluster of orthologous groups (COG) 0116, and most closely resembles the m(2)G10 tRNA methylase Trm11. Phylogenetic analysis reveals a canonical archaeal/bacterial evolutionary separation with 20-30% sequence identities between the two branches, but it is likely that the detailed functions of COG 0116 enzymes differ between the archaeal and bacterial domains. In the archaeal branch, the protein is found exclusively in thermophiles. More distantly related Trm14 homologs were also identified in eukaryotes known to possess the m(2)G6 tRNA modification.

 

Vallee-Belisle, A., et al. (2011). "Transcription Factor Beacons for the Quantitative Detection of DNA Binding Activity." J Am Chem Soc 133(35): 13836-13839.

                The development of convenient, real-time probes for monitoring protein function in biological samples represents an important challenge of the postgenomic era. In response, we introduce here "transcription factor beacons," binding-activated fluorescent DNA probes that signal the presence of specific DNA-binding activities. As a proof of principle, we present beacons for the rapid, sensitive detection of three transcription factors (TATA Binding Protein, Myc-Max, and NF-kappa B), and measure binding activity directly in crude nuclear extracts.

 

Holz-Schietinger, C. and N. O. Reich (2010). "The Inherent Processivity of the Human de Novo Methyltransferase 3A (DNMT3A) Is Enhanced by DNMT3L." Journal of Biological Chemistry 285(38): 29091-29100.

                Human DNMT3A is responsible for de novo DNA cytosine methylation patterning during development. Here we show that DNMT3A methylates 5-8 CpG sites on human promoters before 50% of the initially bound enzyme dissociates from the DNA. Processive methylation is enhanced 3-fold in the presence of DNMT3L, an inactive homolog of DNMT3A, therefore providing a mechanism for the previously described DNMT3L activation of DNMT3A. DNMT3A processivity on human promoters is also regulated by DNA topology, where a 2-fold decrease in processivity was observed on supercoiled DNA in comparison with linear DNA. These results are the first observation that DNMT3A utilizes this mechanism of increasing catalytic efficiency. Processive de novo DNA methylation provides a mechanism that ensures that multiple CpG sites undergo methylation for transcriptional regulation and silencing of newly integrated viral DNA.

 

Pallaoro, A., et al. (2010). "Mapping Local pH in Live Cells Using Encapsulated Fluorescent SERS Nanotags." Small 6(5): 618-622.

               

Purdy, M. M., et al. (2010). "Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion." Archives of Biochemistry and Biophysics 498(1): 13-22.

                The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with mu M affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed. Published by Elsevier Inc.

 

Zhang, F., et al. (2010). "Fabrication of Ag@SiO2@Y2O3:Er Nanostructures for Bioimaging: Tuning of the Upconversion Fluorescence with Silver Nanoparticles." J Am Chem Soc 132(9): 2850-+.

                We demonstrated that the nanostructures comprising silver cores and dense layers of Y2O3:Er separated by a silica shell are an excellent model system to study the interaction between upconversion materials and metals in nanoscale. This architecture allows for versatile control of the Y2O3:Er-metal interaction through control of the silica dielectric spacer thickness and the metal-core size. Finally, the nanoparticles are potentially Interesting as fluorescent labels in. for instance (single particle), imaging experiments or bioassays which require low background or tissue penetrating wavelengths.

 

Bonham, A. J., et al. (2009). "Tracking transcription factor complexes on DNA using total internal reflectance fluorescence protein binding microarrays." Nucleic Acids Research 37(13).

                We have developed a high-throughput protein binding microarray (PBM) assay to systematically investigate transcription regulatory protein complexes binding to DNA with varied specificity and affinity. Our approach is based on the novel coupling of total internal reflectance fluorescence (TIRF) spectroscopy, swellable hydrogel double-stranded DNA microarrays and dye-labeled regulatory proteins, making it possible to determine both equilibrium binding specificities and kinetic rates for multiple protein:DNA interactions in a single experiment. DNA specificities and affinities for the general transcription factors TBP, TFIIA and IIB determined by TIRF-PBM are similar to those determined by traditional methods, while simultaneous measurement of the factors in binary and ternary protein complexes reveals preferred binding combinations. TIRF-PBM provides a novel and extendible platform for multi-protein transcription factor investigation.

 

Braun, G. B., et al. (2009). "Laser-Activated Gene Silencing via Gold Nanoshell-siRNA Conjugates." ACS Nano 3(7): 2007-2015.

                The temporal and spatial control over the delivery of materials such as siRNA into cells remains a significant technical challenge. We demonstrate the pulsed near-infrared (NIR) laser-dependent release of siRNA from coated 40 nm gold nanoshells. Tat-lipid coating mediates the cellular uptake of the nanomaterial at picomolar concentration, while spatiotemporal silencing of a reporter gene (green fluorescence protein) was studied using photomasking. The NIR laser-induced release of siRNA from the nanoshells is found to be power-and time-dependent, through surface-linker bond cleavage, while the escape of the siRNA from endosomes occurs above a critical pulse energy attributed to local heating and cavitation. NIR laser-controlled drug release from functional nanomaterials should facilitate more sophisticated developmental biology and therapeutic studies.

 

Braun, G. B., et al. (2009). "Generalized Approach to SERS-Active Nanomaterials via Controlled Nanoparticle Linking, Polymer Encapsulation, and Small-Molecule Infusion." Journal of Physical Chemistry C 113(31): 13622-13629.

                Over the past decade the emphasis on single-molecule sensitivity of surface-enhanced Raman spectroscopy (SERS) has brought to prominence the special role played by so-called SERS hot spots, often times nanometerscale junctions between nanoparticles (NPs). In this report, molecular linkers are used to mediate the assembly of NPs to dimers and small clusters. When the SERS enhancement is optimized, the aggregation process is quenched by polymer and protein stabilizers that subsequently act as encapsulants resulting in SERS substates with unprecedented enhancement uniformity, reproducibility, and stability. The polymer-stabilized NP junctions were then imprinted with a variety of small molecules that permeated the polymer coat and displaced the linker from the hot spot. The average SERS enhancement of these SERS "nanocapsules" was found to be at least 300x greater than for single NPs, while the Raman/Rayleigh scattering ratio was 101 higher for linked NPs versus nonoptimized aggregates. Single-particle statistics showed that almost every nanocapsule produced intense SERS, suggesting that they are NT dimers and small clusters with the probe molecule resident in a hot spot. Nanocapsules were functionalized and shown to compete successfully with fluorescence imaging in multiplexed identification of cancer cell epitopes at the single-cell and single-nanotag level.

 

Coffin, S. R. and N. O. Reich (2009). "Escherichia coli DNA Adenine Methyltransferase: Intrasite Processivity and Substrate-Induced Dimerization and Activation." Biochemistry 48(31): 7399-7410.

                Methylation of GATC sites in Escherichia coli by DNA adenine methyltransferase (EcoDam) is essential for proper DNA replication timing, gene regulation, and mismatch repair. The low cellular concentration of EcoDam and the high number of GATC sites in the genome (similar to 20000) Support the reliance on methylation efficiency-enhancing strategies such as extensive intersite processivity. Here, we present evidence that EcoDam has evolved other unique mechanisms of activation not commonly observed with restriction-modification methyltransferases, EcoDam dimerizes oil short, synthetic DNA, resulting in enhanced catalysis; however, dimerization is not observed on large genomic DNA where the potential for intersite processive methylation precludes any dimerization-dependent activation. An activated form of the enzyme is apparent on large genomic DNA and can also be achieved with high concentrations of short, synthetic substrates. We suggest that this activation is inherent on polymeric DNA where either multiple GATC sites are available for methylation or the partitioning of the enzyme onto nonspecific DNA is favored. Unlike other restriction-modification methyltransferases, EcoDam carries out intrasite processive catalysis whereby the enzyme-DNA complex methylates both strands of an unmethylated GATC site prior to dissociation From the DNA. This occurs with short 21 bp oligonucleotides and is highly dependent upon salt concentrations. Kinetic modeling which invokes enzyme activation by both dimerization and excess substrate provides mechanistic insights into key regulatory checkpoints for an enzyme involved in multiple, diverse biological pathways.

 

Coffin, S. R. and N. O. Reich (2009). "Escherichia coli DNA Adenine Methyltransferase The Structural Basis of Processive Catalysis and Indirect Read-Out." Journal of Biological Chemistry 284(27): 18390-18400.

                We have investigated the structural basis of processive GATC methylation by the Escherichia coli DNA adenine methyltransferase, which is critical in chromosome replication and mismatch repair. We determined the contribution of the orthologically conserved phosphate interactions involving residues Arg(95), Asn(126), Asn(132), Arg(116), and Lys(139), which directly contact the DNA outside the cognate recognition site (GATC) to processive catalysis, and that of residue Arg(137), which is not conserved and contacts the DNA backbone within the GATC sequence. Alanine substitutions at the conserved positions have large impacts on processivity yet do not impact k(cat)/K(m)(DNA) or DNA affinity (K(D)(DNA)). However, these mutants cause large preferences for GATC sites varying in flanking sequences when considering the pre-steady state efficiency constant k(chem)/K(D)(DNA). These changes occur mainly at the level of the methylation rate constant, which results in the observed decreases in processive catalysis. Thus, processivity and catalytic efficiency (k(cat)/K(m)(DNA)) are uncoupled in these mutants. These results reveal that the binding energy involved in DNA recognition contributes to the assembly of the active site rather than tight binding. Furthermore, the conserved residues (Arg(95), Asn(126), Asn(132), and Arg(116)) repress the modulation of the response of the enzyme to flanking sequence effects. Processivity impacted mutants do not show substrate-induced dimerization as is observed for the wild type enzyme. This study describes the structural means by which an enzyme that does not completely enclose its substrate has evolved to achieve processive catalysis, and how interactions with DNA flanking the recognition site alter this processivity.

 

Estabrook, R. A., et al. (2009). "Coupling Sequence-specific Recognition to DNA Modification." Journal of Biological Chemistry 284(34): 22690-22696.

                Enzymes that modify DNA are faced with significant challenges in specificity for both substrate binding and catalysis. We describe how single hydrogen bonds between M. HhaI, a DNA cytosine methyltransferase, and its DNA substrate regulate the positioning of a peptide loop which is similar to 28 angstrom away. Stopped-flow fluorescence measurements of a tryptophan inserted into the loop provide real-time observations of conformational rearrangements. These long-range interactions that correlate with substrate binding and critically, enzyme turnover, will have broad application to enzyme specificity and drug design for this medically relevant class of enzymes.

 

Koyfman, A. Y., et al. (2009). "Self-Assembly of DNA Arrays into Multilayer Stacks." Langmuir 25(2): 1091-1096.

                We describe the self-assembly of multilayer hexagonal DNA arrays through highly regular interlayer packing. Slow cooling of a mixture of three single-stranded DNA sequences with various Mg(2+) concentrations leads to the self-assembly of diverse multilayer architectures. The self-assembled aggregates were deposited onto mica surfaces and examined with atomic force microscopy. The size of the two-dimensional arrays and subsequent stacking to form multilayer structures are highly dependent on Mg(2+) concentration. DNA bilayers and multilayers of defined shape are favored in 2-5 mM Mg(2+) with an average lateral size of 700 nm. Arrays are much larger (Lip to 20 mu m across) in 10- 15 mM Mg(2+), although multiple layers still make Lip 20-60% of the observed structures. Domains within single layer architectures were identified using Moire pattern analysis. Distinct structural phases within the multilayer assemblies include two layers translated by 17.5 nm and interlayer rotations of 20 degrees and 30 degrees. Three layer assemblies have cubic close packing and taller inultilayer architectures of 2D DNA sheets were also identified.

 

Koyfman, A. Y., et al. (2009). "Cell-Targeted Self-Assembled DNA Nanostructures." J Am Chem Soc 131(40): 14237-+.

                We present two strategies for attaching self-assembled DNA arrays to the surfaces of cells. Our first approach uses biotin-streptavidin interactions to bind DNA architectures to biotinytated cells. The second approach takes advantage of specific antibody-cell surface interactions, conjugated arrays and the subsequent binding to native epidermal growth factor receptors expressed on cancer cells. DNA array-cell surface interactions were visualized by fluorescence, confocal microscopy, and scanning electron microscopy. This novel application of DNA nanoarrays provides strategies to specifically label cell surfaces with micrometer-sized patches, bind cells onto a designed functionalized DNA scaffold, engineer cell/cell networks into microtissues, and deliver materials to cell surfaces.

 

Ricci, F., et al. (2009). "Reagentless, Electrochemical Approach for the Specific Detection of Double- and Single-Stranded DNA Binding Proteins." Anal Chem 81(4): 1608-1614.

                Here we demonstrate a reagentless, electrochemical platform for the specific detection of proteins that bind to single- or double-stranded DNA. The sensor is composed of a double- or single-stranded, redox-tagged DNA probe which is covalently attached to an interrogating electrode. Upon protein binding the current arising from the redox tag is suppressed, indicating the presence of the target. Using this approach we have fabricated sensors against the double-stranded DNA binding proteins TATA-box binding protein and M.HhaI methyltransferase, and against the single-strand binding proteins Escherichia coli SSBP and replication protein A. All four targets are detected at nanomolar concentrations, in minutes, and in a convenient, general, readily reusable, electrochemical format. The approach is specific; we observed no significant cross-reactivity between the sensors. Likewise the approach is selective; it supports, for example, the detection of single strand binding protein directly in crude nuclear extracts. The generality of our approach (including its ability to detect both double- and single-strand binding proteins) and a strong, non-monotonic dependence of signal gain on probe density support a collisional signaling mechanism in which binding alters the collision efficiency, and thus electron transfer efficiency, of the attached redox tag. Given the ubiquity with which protein binding will alter the collisional dynamics of an oligonucleotide, we believe this approach may prove of general utility in the detection of DNA and RNA binding proteins.

 

Zhou, H., et al. (2009). "The Recognition Pathway for the DNA Cytosine Methyltransferase M.HhaI." Biochemistry 48(33): 7807-7816.

                Enzymatic sequence-specific DNA modification involves multiple poorly understood intermediates. DNA methyltransferases like M.HhaI initially bind nonspecific DNA and then selectively bind and modify a unique sequence. High-resolution NMR was used to map conformational changes occurring in M. HhaI upon binding nonspecific DNA, a one base pair altered noncognate DNA sequence, and both hemimethylated and unmethylated cognate DNA sequences. Comparisons with previous NMR studies of the apoenzyme and enzyme-cofactor complex provide snapshots of the pathway to sequence-specific complex formation. Dramatic chemical shift perturbations reaching many distal sites within the protein are detected with cognate DNA, while much smaller changes are observed upon nonspecific and noncognate DNA binding. A cooperative rather than stepwise transition from a nonspecific to a cognate complex is revealed, Furthermore, switching from unmethylated to hemimethylated cognate DNA involves delectable protein conformational changes 20-30 angstrom away from the methyl group, indicating high protein sensitivity and plasticity to DNA modification.

 

Braun, G., et al. (2008). "Enzyme-directed positioning of nanoparticles on large DNA templates." Bioconjug Chem 19(2): 476-479.

                A method to position nanoparticles onto DNA with high resolution using an enzyme-based approach is described. This provides a convenient route to assemble multiple nanoparticles (e.g., An and CdSe) to specific positions with a high level of control and expandability to more complex assemblies. Atomic force microscopy is used to analyze the nanostructures, which have potential interest for biosensor, optical waveguide, molecular electronics, and energy transfer studies.

 

Coffin, S. R. and N. O. Reich (2008). "Modulation of Escherichia coli DNA methyltransferase activity by biologically derived GATC-flanking sequences." Journal of Biological Chemistry 283(29): 20106-20116.

                Escherichia coli DNA adenine methyltransferase (EcoDam) methylates the N-6 position of the adenine in the sequence 5'-GATC-3' and plays vital roles in gene regulation, mismatch repair, and DNA replication. It remains unclear how the small number of critical GATC sites involved in the regulation of replication and gene expression are differentially methylated, whereas the similar to 20,000 GATCs important for mismatch repair and dispersed throughout the genome are extensively methylated. Our prior work, limited to the pap regulon, showed that methylation efficiency is controlled by sequences immediately flanking the GATC sites. We extend these studies to include GATC sites involved in diverse gene regulatory and DNA replication pathways as well as sites previously shown to undergo differential in vivo methylation but whose function remains to be assigned. EcoDam shows no change in affinity with variations in flanking sequences derived from these sources, but methylation kinetics varied 12-fold. A-tracts immediately adjacent to the GATC site contribute significantly to these differences in methylation kinetics. Interestingly, only when the poly(A) is located 5' of the GATC are the changes in methylation kinetics revealed. Preferential methylation is obscured when two GATC sites are positioned on the same DNA molecule, unless both sites are surrounded by large amounts of nonspecific DNA. Thus, facilitated diffusion and sequences immediately flanking target sites contribute to higher order specificity for EcoDam; we suggest that the diverse biological roles of the enzyme are in part regulated by these two factors, which may be important for other enzymes that sequence-specifically modify DNA.

 

Peterson, S. N. and N. O. Reich (2008). "Competitive Lrp and Dam Assembly at the pap Regulatory Region: Implications for Mechanisms of Epigenetic Regulation." Journal of Molecular Biology 383(1): 92-105.

                Escherichia coli DNA adenine methyltransferase (Dam) and Leucine-responsive regulatory protein (Lrp) are key regulators of the pap operon, which codes for the pilus proteins necessary for uropathogenic E. coli cellular adhesion. Pie pap operon is regulated by a phase variation mechanism in which the methylation states of two GATC sites in the pap regulatory region and the binding position of Lrp determine whether the pilus genes are expressed. The post-replicative reassembly of Dam, Lip, and the local regulator PapI onto a hemimethylated pap intermediate is a critical step of the phase variation switching mechanism and is not well understood. We show that Lrp, in the presence and in the absence of PapI and nonspecific DNA, specifically protects pap regulatory GATC sites from Dam methylation when allowed to compete with Dam for assembly on unmethylated and hemimethylated pap DNA. The methylation protection is dependent upon the concentration of Lrp and does not occur with non-regulatory GATC sites. Our data suggest that only at low Lrp concentrations will Dam compete effectively for binding and methylation of the proximal GATC site, leading to a phase switch resulting in the expression of pili. (C) 2008 Elsevier Ltd. All riahts reserved.

 

Wilkinson, S., et al. (2008). "Molecular scale architecture: Engineered three- and four-way junctions." Bioconjug Chem 19(2): 470-475.

                Biomolecular self-assembly provides a basis for the bottom-up construction of useful and diverse nanoscale architectures. DNA is commonly used to create these assemblies and is often exploited as a lattice or an array. Although geometrically rigid and highly predictable, these sheets of repetitive constructs often lack the ability to be enzymatically manipulated or elongated by standard biochemical techniques. Here, we describe two approaches for. the construction of position-controlled, molecular-scale, discrete, three- and four-way DNA junctions. The first approach for constructing these junctions relies on the use of nonmigrating cruciforms generated from synthetic oligonucleotides to which large, biologically generated, double-stranded DNA segments are enzymatically ligated. The second approach utilitizes the DNA methyltransferase-based SMILing (sequence-specific methyltransferase-induced labeling of DNA) method to site-specifically incorporate a biotin within biologically derived DNA. Streptavidin is then used to form junctions between unique DNA strands. The resultant assemblies have precise and predetermined connections with lengths that can be varied by enzymatic or hybridization techniques, or geometrically controlled with standard DNA functionalization methods. These junctions are positioned with single nucleotide resolution on large, micrometer-length templates. Both approaches generate DNA assemblies which are fully compatible with standard recombinant methods and thus provide a novel basis for nanoengineering applications.

 

Youngblood, B. and N. O. Reich (2008). "The early expressed HIV-1 genes regulate DNMT1 expression." Epigenetics 3(3): 149-156.

                DNA methylation-mediated transcriptional regulation is essential for human development and diverse diseases ensue when this process is dysregulated. Viruses, including HIV-1, alter the expression of human DNA methyltransferases (DNMTs) and various T cell-specific genes. Here we show that HIV-1 induction of DNMT1 results from overexpression of the early expressed HIV-1 proteins, and this induction is prevented with resveratrol which interferes with the transcription factor AP1 pathway. The HIV-1 responsive element resides in the 5' most 420 bp of the -1634 to +71 DNMT1 promoter; positioning of this truncated promoter proximal to a hybrid SV40-DNMT1 reporter results in HIV-1-dependent regulation. HIV-1 mediated induction of DNMT1 is not specific to T-cells, and does not require receptor-mediated endocytosis. The characterization of HIV-1 mediated DNMT1 regulation provides a basis for identifying viral and cellular factors necessary for the de novo DNA methylation of cellular genes.

 

Youngblood, B., et al. (2008). "Differential stabilization of reaction intermediates: specificity checkpoints for M.EcoRI revealed by transient fluorescence and fluorescence lifetime studies." Nucleic Acids Research 36(9): 2917-2925.

                M.EcoRI, a bacterial sequence-specific S-adenosyl-L-methionine-dependent DNA methyltransferase, relies on a complex conformational mechanism to achieve its remarkable specificity, including DNA bending, base flipping and intercalation into the DNA. Using transient fluorescence and fluorescence lifetime studies with cognate and noncognate DNA, we have characterized several reaction intermediates involving the WT enzyme. Similar studies with a bending-impaired, enhanced-specificity M.EcoRI mutant show minimal differences with the cognate DNA, but significant differences with noncognate DNA. These results provide a plausible explanation of the way in which destabilization of reaction intermediates can lead to changes in substrate specificity.

 

Bonham, A. J., et al. (2007). "Detection of sequence-specific protein-DNA interactions via surface enhanced resonance Raman scattering." J Am Chem Soc 129(47): 14572-+.

                We developed a new strategy to detect protein-DNA binding through surface enhanced resonance Raman scattering (SERRS). Silver-plated DNA and gold nanoparticle assemblies were used to identify sequence-specific and concentration-dependent binding of a DNA cytosine-C5-methyltransferase and the eukaryotic transcriptional regulator, TATA binding protein. Proteins were identified through specific Raman-active labels, and affinities observed correlate well with those determined by other methods. Raman-active labeling and interchangeable DNA sequences create a platform for versatile investigation of multiprotein complexes.

 

Braun, G., et al. (2007). "Chemically patterned microspheres for controlled nanoparticle assembly in the construction of SERS hot spots." J Am Chem Soc 129(25): 7760-+.

                An important challenge in developing ultra-sensitive surface enhanced Raman spectroscopy (SERS) platforms lies in the creation of nanoscale hot spots, locations where the electromagnetic field is greatly concentrated. Even when this is successful, finding these hot spots is a difficult task. We describe a novel chemical microsphere patterning technique utilizing contact masking during silanization for bonding silver nanoparticles preferentially to geometrically restricted sites. Small bifunctional linkers (i.e., < 2 nm) are used to bind silver particles on different microspheres drawing the two together, thereby forming small nanoparticle aggregates containing one or more hot spots with the dimensions of the linker at the junction. The microspheres limit the extent of nanoparticle aggregation and are large enough to be visible by optical microscopy leading in most cases directly to the location of the hot spots and hence the most intense SERS signals.

 

Fabris, L., et al. (2007). "A heterogeneous PNA-based SERS method for DNA detection." J Am Chem Soc 129(19): 6086-+.

                A simple method for ssDNA detection based on SERS signals and PNA slides is reported. Upon hybridization with ssDNA, the surface charge of the PNA slides changes from neutral to negative. Subsequent treatment with partly aggregated, positively charged silver nanoparticles results in selective electrostatic adsorption onto surfaces containing PNA/ssDNA duplexes. Addition of rhodamine6G gives rise to SERS signals characteristic of this dye, which are diagnostic of the hybridization event. Characterization by SERS maps and AFM reveals that the distribution of nanoparticles is random and that approximately 1 in 10 aggregate sites is responsible for the SERS spectra.

 

Kim, J. H., et al. (2007). "Specific and sensitive detection of nucleic acids and RNases using gold nanoparticle-RNA-fluorescent dye conjugates." Chemical Communications(42): 4342-4344.

                Gold nanoparticles were modified with RNA and utilized to detect specific DNA sequences and various RNA nucleases.

 

Peterson, S. N., et al. (2007). "The role of high affinity non-specific DNA binding by Lrp in transcriptional regulation and DNA organization." Journal of Molecular Biology 369(5): 1307-1317.

                Transcriptional regulatory proteins typically bind specific DNA sequences with similar to 10(3)-10(7)-fold higher affinity than non-specific DNA and this discrimination is essential for their in vivo function. Here we show that the bacterial leucine-responsive regulatory protein (Lrp) does not follow this trend and has a similar to 20-400-fold binding discrimination between specific and non-specific DNA sequences. We suggest that the dual function of Lrp to regulate genes and to organize DNA utilizes this unique property. A similar to 20-fold decrease in binding affinity from specific DNA is dependent upon cryptic binding sites, including the sequence GN(2-3)TTT and A-tracts. Removal of these sites still results in high binding affinity, only similar to 70-fold weaker than that of specific sites. Similar to Lrp's binding of specific sites in the pap and ilvIH promoters, Lrp binds cooperatively to non-specific DNA; thus, protein/protein interactions are important for both specific and nonspecific DNA binding. When considering this cooperativity of Lrp binding, the binding selectivity to specific sites may increase to a maximum of similar to 400-fold. Neither leucine nor the pap-specific local regulator Papl alter Lrp's nonspecific binding affinity or cooperative binding of non-specific DNA. We hypothesize that Lrp combines low sequence discrimination and relatively high intracellular protein concentrations to ensure its ability to regulate the transcription of specific genes while also functioning as a nucleoid-associated protein. Modeling of Lrp binding data and comparison to other proteins with regulatory and nucleoid-associated properties suggests similar mechanisms. Published by Elsevier Ltd.

 

Shieh, F.-K. and N. O. Reich (2007). "AdoMet-dependent methyl-transfer: Glu(119) is essential for DNA c5-cytosine methyltransferase M.Hhal." Journal of Molecular Biology 373(5): 1157-1168.

                The role of Glu119 in S-adenosyl-L-methionine-dependent DNA methyltransferase M.HhaI-catalyzed DNA methylation was studied. Glu119 belongs to the highly conserved Glu/Asn/Val motif found in all DNA C5-cytosine methyltransferases, and its importance for M.HhaI function remains untested. We show that formation of the covalent intermediate between Cys81 and the target cytosine requires Glu119, since conversion to Ala, Asp or Gln lowers the rate of methyl transfer 10(2)-10(6) fold. Further, unlike the wild-type M.HhaI, these mutants are not trapped by the substrate in which the target cytosine is replaced with the mechanism-based inhibitor 5-fluorocytosine. The DNA binding affinity for the Glu119Asp mutant is decreased 10(3)-fold. Thus, the ability of the enzyme to stabilize the extrahelical cytosine is coupled directly to tight DNA binding. The structures of the ternary protein/DNA/ AdoHcy complexes for both the Glu119Ala and Glu119Gln mutants (2.70 angstrom and 2.75 angstrom, respectively) show that the flipped base is positioned nearly identically with that observed in the wild-type M.HhaI complex. A single water molecule in the Glu119Ala structure between Ala119 and the extrahelical cytosine N3 is lacking in the Glu119Gln and wild-type M.HhaI structures, and most likely accounts for this mutant's partial activity. Glu119 has essential roles in activating the target cytosine for nucleophilic attack and contributes to tight DNA binding. (C) 2007 Elsevier Ltd. All rights reserved.

 

Wood, D. K., et al. (2007). "A feasible approach to all-electronic digital labeling and readout for cell identification." Lab on a Chip 7(4): 469-474.

                We present two critical innovations that enable a unique, purely electronic approach to microfluidic whole-cell analysis, focusing on the problem of cell identification and sorting. We used fully-scalable lithographic techniques to microfabricate digital barcodes, providing a means for low-cost, large volume production. We have demonstrated molecular functionalization of the barcodes, using biotin-streptavidin, as well as human CD4 antibody, and we have successfully linked the barcodes to polystyrene beads using the biotin-streptavidin complex. This functionalization allows unique barcodes to be attached to specific cell types, based on phenotype. We have also implemented an electronic barcode readout scheme, using a radio frequency microsensor integrated in an elastomeric microfluidic channel, that can read individual barcodes at rates in excess of 1000 labels s(-1). The barcodes are biologically compatible, and coupled with the electronic sensing technology, provide a route to compact, inexpensive, disposable cell identification, sorting and purification.

 

Wu, K.-L., et al. (2007). "Facile synthesis of naphthoquinone spiroketals by diastereoselective oxidative 3+2 cycloaddition." Organic Letters 9(26): 5537-5540.

                A highly selective oxidative [3 + 2] cycloaddition of chiral enol ethers and hydroxynaphthoquinone is described. This convergent strategy is amenable to an enantioselective synthesis of beta-rubromycin and related naphthoquinone spiroketals. Several compounds were found to inhibit DNA-polymerase and telomerase in a manner resembling a-rubromycin and beta-rubromycin.

 

Youngblood, B., et al. (2007). "S-Adenosyl-L-methionine-dependent methyl transfer: Observable precatalytic intermediates during DNA cytosine methylation." Biochemistry 46(30): 8766-8775.

                S-Adenosyl-< SCP > l </SCP >-methionine- (AdoMet-) dependent methyltransferases are widespread, play critical roles in diverse biological pathways, and are antibiotic and cancer drug targets. Presently missing from our understanding of any AdoMet-dependent methyl-transfer reaction is a high-resolution structure of a precatalytic enzyme/AdoMet/DNA complex. The catalytic mechanism of DNA cytosine methylation was studied by structurally and functionally characterizing several active site mutants of the bacterial enzyme M.HhaI. The 2.64 A resolution protein/DNA/AdoMet structure of the inactive C81A M.HhaI mutant suggests that active site water, a similar to 13 degrees tilt of the target base toward the active site nucleophile, and the presence or absence of the cofactor methylsulfonium are coupled via a hydrogen-bonding network involving Tyr167. The active site in the mutant complex is assembled to optimally align the pyrimidine for nucleophilic attack and subsequent methyl transfer, consistent with previous molecular dynamics ab initio and quantum mechanics/molecular mechanics calculations. The mutant/DNA/AdoHcy structure (2.88 A resolution) provides a direct comparison to the postcatalytic complex. A third C81A ternary structure (2.22 A resolution) reveals hydrolysis of AdoMet to adenosine in the active site, further validating the coupling between the methionine portion of AdoMet and ultimately validating the structural observation of a prechemistry/postchemistry water network. Disruption of this hydrogen-bonding network by a Tyr167 to Phe167 mutation does not alter the kinetics of nucleophilic attack or methyl transfer. However, the Y167F mutant shows detectable changes in k(cat), caused by the perturbed kinetics of AdoHcy release. These results provide a basis for including an extensive hydrogen-bonding network in controlling the rate-limiting product release steps during cytosine methylation.

 

Zhou, H., et al. (2007). "Long-range structural and dynamical changes induced by cofactor binding in DNA methyltransferase M.HhaI." Biochemistry 46(24): 7261-7268.

                The bacterial DNA cytosine methyltransferase M.HhaI sequence-specifically modifies DNA in an S-adenosylmethionine dependent reaction. The enzyme stabilizes the target cytosine (G (C) under bar GC) into an extrahelical position, with a concomitant large movement of an active site loop involving residues 80-99. We used multidimensional, transverse relaxation-optimized NMR experiments to assign nearly 80% of all residues in the cofactor-bound enzyme form, providing a basis for detailed structural and dynamical characterization. We examined details of the previously unknown effects of the cofactor binding with M.HhaI in solution. Addition of the cofactor results in numerous structural changes throughout the protein, including those decorating the cofactor binding site, and distal residues more than 30 A away. The active site loop is involved in motions both on a picosecond to nanosecond time scale and on a microsecond to millisecond time scale and is not significantly affected by cofactor binding except for a few N-terminal residues. The cofactor also affects residues near the DNA binding cleft, suggesting a role for the cofactor in regulating DNA interactions. The allosteric properties we observed appear to be closely related to the significant amount of dynamics and dynamical changes in response to ligand binding detected in the protein.

 

Mashhoon, N., et al. (2006). "Selective inhibitors of bacterial DNA adenine methyltransferases." Journal of Biomolecular Screening 11(5): 497-510.

                The authors describe the discovery and characterization of several structural classes of small-molecule inhibitors of bacterial DNA adenine methyltransferases. These enzymes are essential for bacterial virulence (DNA adenine methyltransferase [DAM]) and cell viability (cell cycle-regulated methyltransferase [CcrM]). Using a novel high-throughput fluorescence-based assay and recombinant DAM and CcrM, the authors screened a diverse chemical library. They identified 5 major structural classes of inhibitors composed of more than 350 compounds: cyclopentaquinolines, phenyl vinyl furans, pyrimidine-diones, thiazolidine-4-ones, and phenyl-pyrroles. DNA binding assays were used to identify compounds that interact directly with DNA. Potent compounds selective for the bacterial target were identified, whereas other compounds showed greater selectivity for the mammalian DNA cytosine methyltransferase, Dnmt1. Enzyme inhibition analysis identified mechanistically distinct compounds that interfered with DNA or cofactor binding. Selected compounds demonstrated cell-based efficacy. These small-molecule DNA methyltransferase inhibitors provide useful reagents to probe the role of DNA methylation and may form the basis of developing novel antibiotics.

 

Peterson, S. N. and N. O. Reich (2006). "GATC flanking sequences regulate dam activity: Evidence for how Dam specificity may influence pap expression." Journal of Molecular Biology 355(3): 459-472.

                Escherichia coli DNA adenine methyltransferase (Dam) plays essential roles in DNA replication, mismatch repair and gene regulation. The differential methylation by Dam of the two GATC sequences in the pap promoter regulates the expression of pili genes necessary for uropathogenic E. coli cellular adhesion. Dam processively methylates GATC sites in various DNA substrates, yet the two pap GATC sites are not processively methylated. We previously proposed that the flanking sequences surrounding the two pap GATC sites contribute to the enzyme's distributive methylation. We show here that replacement of the poorly methylated pap GATC sites with sites predicted to be processively methylated indeed results in an increase in Dam processivity. The increased processivity is due to a change in the methyltransfer kinetics and not the binding efficiency of Dam. A competition experiment in which the flanking sequences of only one pap GATC site were altered demonstrates that the GATC flanking sequences directly regulate the enzyme's catalytic efficiency. The GATC flanking sequences in Dam-regulated promoters in E. coli and other bacteria are similar to those in the pap promoter. Gene regulation from some of these promoters involves mechanisms and proteins that are quite different from those in the pap operon. Further, GATC sequences previously identified to remain unmethylated within the E. coli genome, but whose function remains largely unassigned, are flanked by sequences predicted to be poorly methylated. We conclude that the GATC flanking sequences may be critical for expression of pap and other Dam-regulated genes by affecting the activity of Dam at such sites and, thus, its processivity. A model is proposed, illustrating how the sequences flanking the GATC sites in Dam-regulated promoters may contribute to this epigenetic mechanism of gene expression, and how flanking sequences contribute to the diverse biological roles of Dam. (c) 2005 Elsevier Ltd. All rights reserved.

 

Purdy, M. M. and N. O. Reich (2006). "Modulation of mammalian DNA methyltransferase activity by RNA." Abstracts of Papers of the American Chemical Society 232: 778-778.

               

Purdy, M. M. and N. O. Reich (2006). "DNA binding by HhaI DNA methyltransferase domain interface mutants." Faseb Journal 20(5): A901-A901.

               

Reich, N. O. (2006). "The allosteric regulation of Dnmt1 catalysis and processivity by nucleic acids." Faseb Journal 20(4): A77-A77.

               

Shieh, F.-K., et al. (2006). "The role of Arg165 towards base flipping, base stabilization and catalysis in M.Hhal." Journal of Molecular Biology 362(3): 516-527.

                Arg165 forms part of a previously identified base flipping motif in the bacterial DNA cytosine methyltransferase, M.HhaI. Replacement of Arg165 with Ala has no detectable effect on either DNA or AdoMet affinity, yet causes the base flipping and restacking transitions to be decreased similar to 16 and 190-fold respectively, thus confirming the importance of this motif. However, these kinetic changes cannot account for the mutant's observed 9 10(5)-fold decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure (2.79 angstrom resolution) shows the target cytosine to be positioned similar to 30 degrees into the major groove, which is consistent with a major groove pathway for nucleotide flipping. The pyrimidine-sugar chi angle is rotated to approximately +171 degrees, from a range of -95 degrees to -120 degrees in B DNA, and -77 degrees in the WT M.HhaI complex. Thus, Arg165 is important for maintaining the cytosine positioned for nucleophilic attack by Cys81. The cytosine sugar pucker is in the C2'-endo-C3'-exo (South conformation), in contrast to the previously reported C3'-endo (North conformation) described for the original 2.70 A resolution cocrystal structure of the WT M.HhaI/DNA complex. We determined a high resolution structure of the WT M.Hhal/DNA complex (1.96 angstrom) to better determine the sugar pucker. This new structure is similar to the original, lower resolution WT M.HhaI complex, but shows that the sugar pucker is O4'-endo (East conformation), intermediate between the South and North conformers. In summary, Arg165 plays significant roles in base flipping, cytosine positioning, and catalysis. Furthermore, the previously proposed M.HhaI-mediated changes in sugar pucker may not be an important contributor to the base flipping mechanism. These results provide insights into the base flipping and catalytic mechanisms for bacterial and eukaryotic DNA methyltransferases. (c) 2006 Elsevier Ltd. All rights reserved.

 

Wilkinson, S. R. and N. O. Reich (2006). "Nanoparticle based detection of multiprotein complexes on DNA." Faseb Journal 20(4): A101-A101.

               

Wu, H., et al. (2006). "Inactivation of DNA adenine methyltransferase alters virulence factors in Actinobacillus actinomycetemcomitans." Oral Microbiology and Immunology 21(4): 238-244.

                DNA adenine methyltransferase (DAM) plays critical roles in diverse biological pathways in gram-negative bacteria, and specifically in regulating the expression of virulence genes in several organisms. Actinobacillus actinomycetemcomitans plays an important role in the pathogenesis of juvenile and adult periodontal disease, yet little is known about its mechanisms of gene regulation. DAM is shown here to directly or indirectly affect well-known A. actinomycetemcomitans virulence factors. A mutant A. actinomycetemcomitans strain lacking the dam gene was created by homologous recombination and shows normal growth phenotypes when grown exponentially. This mutant strain has four sixfold increased levels of extracellular leukotoxin, altered cellular levels of leukotoxin, and significant changes in bacterial invasion of KB oral epithelial cells. These results provide a basis for further characterization of regulatory mechanisms that control A. actinomycetemcomitans virulence.

 

Youngblood, B. and N. O. Reich (2006). "Conformational transitions as determinants of specificity for the DNA methyltransferase EcoRI." Journal of Biological Chemistry 281(37): 26821-26831.

                Changes in DNA bending and base flipping in a previously characterized specificity-enhanced M. EcoRI DNA adenine methyltransferase mutant suggest a close relationship between precatalytic conformational transitions and specificity (Allan, B. W., Garcia, R., Maegley, K., Mort, J., Wong, D., Lindstrom, W., Beechem, J. M., and Reich, N. O. (1999) J. Biol. Chem. 274, 19269-19275). The direct measurement of the kinetic rate constants for DNA bending, intercalation, and base flipping with cognate and noncognate substrates (GAATTT, GGATTC) of wild type M. EcoRI using fluorescence resonance energy transfer and 2-aminopurine fluorescence studies reveals that DNA bending precedes both intercalation and base flipping, and base flipping precedes intercalation. Destabilization of these intermediates provides a molecular basis for understanding how conformational transitions contribute to specificity. The 3500- and 23,000-fold decreases in sequence specificity for noncognate sites GAATTT and GGATTC are accounted for largely by an similar to 2500-fold increase in the reverse rate constants for intercalation and base flipping, respectively. Thus, a predominant contribution to specificity is a partitioning of enzyme intermediates away from the Michaelis complex prior to catalysis. Our results provide a basis for understanding enzyme specificity and, in particular, sequence-specific DNA modification. Because many DNA methyltransferases and DNA repair enzymes induce similar DNA distortions, these results are likely to be broadly relevant.

 

Youngblood, B., et al. (2006). "Determinants of sequence-specific DNA methylation: Target recognition and catalysis are coupled in M.HhaI." Biochemistry 45(51): 15563-15572.

                Sequence specificity studies of the wild-type bacterial DNA cytosine C-5 methyltransferase HhaI were carried out with cognate (5'GCGC3') and noncognate DNA substrates containing single base pair changes at the first and the fourth position (underlined). Specificity for noncognate site methylation at the level of k(cat)/K-D(DNA) is decreased 9000-80000-fold relative to the cognate site, manifested through changes in methylation, or a prior step, and changes in K-D(DNA). Analysis of a new high-resolution enzyme-DNA cocrystal structure provides a partial mechanistic understanding of this discrimination. To probe the significance of conformational transitions occurring prior to catalysis in determining specificity, we analyzed the double mutant (H127A/T132A). These amino acid substitutions disrupt the interface between the flexible loop (residues 80-99), which interacts with the DNA minor groove, and the active site. The mutant's methylation of the cognate site is essentially unchanged, yet its methylation of noncognate sites is decreased up to 460-fold relative to the wild-type enzyme. We suggest that a significant contribution to M.HhaI's specificity involves the stabilization of reaction intermediates prior to methyl transfer, mediated by DNA minor groove-protein flexible loop interactions.

 

Youngblood, B., et al. (2006). "Engineered extrahelical base destabilization enhances sequence discrimination of DNA methyltransferase M.HhaI." Journal of Molecular Biology 362(2): 334-346.

                Improved sequence specificity of the DNA cytosine methyltransferase HhaI was achieved by disrupting interactions at a hydrophobic interface between the active site of the enzyme and a highly conserved flexible loop. Transient fluorescence experiments show that mutations disrupting this interface destabilize the positioning of the extrahelical, "flipped" cytosine base within the active site. The ternary crystal structure of the F124A M.HhaI bound to cognate DNA and the cofactor analogue S-adenosyl-L-homocysteine shows an increase in cavity volume between the flexible loop and the core of the enzyme. This cavity disrupts the interface between the loop and the active site, thereby destabilizing the extrahelical target base. The favored partitioning of the base-flipped enzyme-DNA complex back to the base-stacked intermediate results in the mutant enzyme discriminating better than the wild-type enzyme against non-cognate sites. Building upon the concepts of kinetic proofreading and our understanding of M.HhaI, we describe how a 16-fold specificity enhancement achieved with a double mutation at the loop/active site interface is acquired through destabilization of intermediates prior to methyltransfer rather than disruption of direct interactions between the enzyme and the substrate for M.HhaI. (c) 2006 Elsevier Ltd. All rights reserved.

 

Braun, G., et al. (2005). "Gold nanoparticle decoration of DNA on silicon." Langmuir 21(23): 10699-10701.

                Electrostatic assembly of cationic nanoparticles onto the negatively charged backbone of double-stranded DNA has been shown to produce one-dimensional chains with potential use as nanoelectronic components. In this paper, micron long DNA templates stretched on aminosilane- and hexamethyldisilazane-modified silicon surfaces are used to assemble 3.5 nm gold nanoparticles passivated with cationic thiocholine. Atomic force microscopy is used to analyze the density and defects along the similar to 5 nm high structures, with comparison between positively charged and neutral surfaces. Low background adsorption of nanoparticles is facilitated by both these surface chemistries, while the neutral surface yields a more densely packed assembly.

 

Estabrook, R. A., et al. (2005). "Statistical colevolution analysis and molecular dynamics: Identification of amino acid pairs essential for catalysis." Proc Natl Acad Sci U S A 102(4): 994-999.

                Molecular dynamics (MD) simulations of Hhal DNA methyltransferase and statistical coupling analysis (SCA) data on the DNA cytosine methyltransferase family were combined to identify residues that are coupled by coevolution and motion. The highest ranking correlated pairs from the data matrix product (SCA(.)MD) are colocalized and form stabilizing interactions; the anticorrelated pairs are separated on average by 30 A and form a clear focal point centered near the active site. We suggest that these distal anticorrelated pairs are involved in mediating active-site compressions that may be important for catalysis. Mutants that disrupt the implicated interactions support the validity of our combined SCA(.)MD approach.

 

Koyfman, A. Y., et al. (2005). "Controlled spacing of cationic gold nanoparticles by nanocrown RNA." J Am Chem Soc 127(34): 11886-11887.

               

Oza, J. P., et al. (2005). "DNA methylation modulates Salmonella enterica serovar Typhimurium virulence in Caenorhabditis elegans." Fems Microbiology Letters 245(1): 53-59.

                Salmonella enterica serovar Typhimurium was previously shown to be virulent in Caenorhabditis elegans. Here we demonstrate that DNA adenine methyltransferase (DAM) modulates Salmonella virulence in the nematode, as it does in mice. After 5 days of continual exposure to bacteria, twice as many worms died when exposed to the wild-type than the dam-mutant strain of Salmonella. Similar trends in virulence were observed when worms were exposed to Salmonella strains for 5 h and transferred to the avirulent Escherichia coli OP50. While a 10-fold attenuation was observed in the absence of DAM, the darn-strain was still able to infect and persist in the host worm. Our results further support the use of C elegans as an accessible and readily studied animal model of bacterial pathogenesis. However, our results suggest that crucial host responses differ between the murine and nematode models. Additionally, we carried out preliminary liquid culture based experiments with the long term goal of developing high throughput animal based screens of DAM inhibitors. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

 

Purdy, M. M. and N. O. Reich (2005). "DNA binding and hemimethylation preference of HhaI DNA methyltransferase domain interface mutants." Abstracts of Papers of the American Chemical Society 230: U626-U626.

               

Svedruzic, Z. M. and N. O. Reich (2005). "Mechanism of allosteric regulation of Dnmt1's processivity." Biochemistry 44(45): 14977-14988.

                We have analyzed the relationship between the allosteric regulation and processive catalysis of DNA methyltransferase 1 (Dnmt1). Processivity is described quantitatively in terms of turnover rate, DNA dissociation rate, and processivity probability. Our results provide further evidence that the active site and the allosteric sites on Dnmt1 can bind DNA independently. Dnmt1's processive catalysis on unmethylated DNA is partially inhibited when the allosteric site binds unmethylated DNA and fully inhibited when the allosteric site binds a single-stranded oligonucleotide inhibitor. The partial inhibition by unmethylated DNA is caused by a decrease in the turnover rate and an increase in the substrate DNA dissociation rate. Processive catalysis with premethylated DNA is not affected if the allosteric site is exposed to premethylated DNA but is fully inhibited if the allosteric site binds unmethylated DNA or poly(dA-dT). In sum, the occupancy of the allosteric site modulates the enzyme's commitment to catalysis, which reflects the nature of the substrate and the DNA bound at the allosteric site. Our in vitro results are consistent with the possibility that the processive action of Dnmt1 may be regulated in vivo by specific regulatory nucleic acids such as DNA, RNA, or poly(ADP-ribose).

 

Svedruzic, Z. M. and N. O. Reich (2005). "DNA cytosine C-5 methyltransferase Dnmt1: Catalysis-dependent release of allosteric inhibition." Biochemistry 44(27): 9472-9485.

                We followed the cytosine C-5 exchange reaction with Dnmt1 to characterize its preference for different DNA substrates, its allosteric regulation, and to provide a basis for comparison with the bacterial enzymes. We determined that the methyl transfer is rate-limiting, and steps up to and including the cysteine-cytosine covalent intermediate are in rapid equilibrium. Changes in these rapid equilibrium steps account for many of the previously described features of Dnmt1 catalysis and specificity including faster reactions with premethylated DNA versus unmethylated DNA, faster reactions with DNA in which guanine is replaced with inosine [poly(dC-dG) vs poly(dI-dC)], and 10-100-fold slower catalytic rates with Dnmt1 relative to the bacterial enzyme M.HhaI. Dnmt1 interactions with the guanine within the CpG recognition site can prevent the premature release of the target base and solvent access to the active site that could lead to mutagenic deamination. Our results suggest that the beta-elimination step following methyl transfer is not mediated by free solvent. Dnmt1 shows a kinetic lag in product formation and allosteric inhibition with unmethylated DNA that is not observed with premethylated DNA. Thus, we suggest the enzyme undergoes a slow relief from allosteric inhibition upon initiation of catalysis on unmethylated DNA. Notably, this relief from allosteric inhibition is not caused by self-activation through the initial methylation reaction, as the same effect is observed during the cytosine C5 exchange reaction in the absence of AdoMet. We describe limitations in the Michaelis-Menten kinetic analysis of Dnmt1 and suggest alternative approaches.

 

Yun, C. S., et al. (2005). "Nanometal surface energy transfer in optical rulers, breaking the FRET barrier." J Am Chem Soc 127(9): 3115-3119.

                Optical-based distance measurements are essential for tracking biomolecular conformational changes, drug discovery, and cell biology. Traditional Forster resonance energy transfer (FRET) is efficient for separation distances up to 100 A. We report the first successful application of a dipole-surface type energy transfer from a molecular dipole to a nanometal surface that more than doubles the traditional Forster range (220 Angstrom) and follows a 1/R(4) distance dependence. We appended a 1.4 nm Au cluster to the 5' end of one DNA strand as the energy acceptor and a fluorescein (FAM) to the 5' end of the complementary strand as the energy donor. Analysis of the energy transfer on DNA lengths (15, 20, 30, 60bp), complemented by protein-induced DNA bending, provides the basis for fully mapping the extent of this dipole surface type mechanism over its entire usable range (50-250 Angstrom). Further, protein function is fully compatible with these nanometal-DNA constructs. Significantly extending the range of optical based methods in molecular rulers is an important leap forward for biophysics.

 

Hopkins, B. B. and N. O. Reich (2004). "Simultaneous DNA binding, bending, and base flipping - Evidence for a novel M. EcoRI methyltransferase-DNA complex." Journal of Biological Chemistry 279(35): 37049-37060.

                We measured the kinetics of DNA bending by M. EcoRI using DNA labeled at both 5'-ends and observed changes in fluorescence resonance energy transfer. Although known to bend its cognate DNA site, energy transfer is decreased upon enzyme binding. This unanticipated effect is shown to be robust because we observe the identical decrease with different dye pairs, when the dye pairs are placed on the respective 3'-ends, the effect is cofactor- and protein-dependent, and the effect is observed with duplexes ranging from 14 through 17 base pairs. The same labeled DNA shows the anticipated increased energy transfer with EcoRV endonuclease, which also bends this sequence, and no change in energy transfer with EcoRI endonuclease, which leaves this sequence unbent. We interpret these results as evidence for an increased end-to-end distance resulting from M. EcoRI binding, mediated by a mechanism novel for DNA methyltransferases, combining DNA bending and an overall expansion of the DNA duplex. The M. EcoRI protein sequence is poorly accommodated into well defined classes of DNA methyltransferases, both at the level of individual motifs and overall alignment. Interestingly, M. EcoRI has an intercalation motif observed in the FPG DNA glycosylase family of repair enzymes. Enzyme-dependent changes in anisotropy and fluorescence resonance energy transfer have similar rate constants, which are similar to the previously determined rate constant for base flipping; thus, the three processes are nearly coincidental. Similar fluorescence resonance energy transfer experiments following AdoMet-dependent catalysis show that the unbending transition determines the steady state product release kinetics.

 

Jennings, T. L., et al. (2004). "Bio-nanomaterials as molecular rulers: Application of nanoparticles for distance measurements in proteo-nucleic systems." Abstracts of Papers of the American Chemical Society 228: U897-U897.

               

Svedruzic, Z. M. and N. O. Reich (2004). "The mechanism of target base attack in DNA cytosine carbon 5 methylation." Biochemistry 43(36): 11460-11473.

                We measured the tritium exchange reaction on cytosine C-5 in the presence of AdoMet analogues to investigate the catalytic mechanism of the bacterial DNA cytosine methyltransferase M.HhaI. Poly(dG-dC) and poly(dI-dC) substrates were used to investigate the function of the active site loop (residues 80-99), stability of the extrahelical base, base flipping mechanism, and processivity on DNA substrates. On the basis of several experimental approaches, we show that methyl transfer is the rate-limiting pre-steady-state step. Further, we show that the active site loop opening contributes to the rate-limiting step during multiple cycles of catalysis. Target base activation and nucleophilic attack by cysteine 81 are fast and readily reversible. Thus, the reaction intermediates involving the activated target base and the extrahelical base are in equilibrium and accumulate prior to the slow methyl transfer step. The stability of the activated target base depends on the active site loop closure, which is dependent on the hydrogen bond between isoleucine 86 and the guanine 5' to the target cytosine. These interactions prevent the premature release of the extrahelical base and uncontrolled solvent access; the latter modulates the exchange reaction and, by implication, the mutagenic deamination reaction. The processive catalysis by M.HhaI is also regulated by the interaction between isoleucine 86 and the DNA substrate. Nucleophilic attack by cysteine 81 is partially rate limiting when the target base is not fully stabilized in the extrahelical position, as observed during the reaction with the Gln(237)Trp mutant or in the cytosine C-5 exchange reaction in the absence of the cofactor.

 

Aubol, B. E. and N. O. Reich (2003). "Murine DNA cytosine C-5-methyltransferase: in vitro studies of de novo methylation spreading." Biochem Biophys Res Commun 310(1): 209-214.

                The preference of murine DNA (cytosine-5)-methyltransferase (Dnmt1) for single stranded DNA substrates is increased up to 50-fold by the presence of a proximal 5-methyl cytosine (5(me)C). This modulation is distance-dependent and is due to an enhanced binding affinity and minor changes in catalytic efficiency. No modulation was observed with double stranded DNA. Modulation requires that the 5(me)C moiety be attached to the DNA strand containing the CpG methylation target. Our results support a model in which 5(me)C binding by the enzyme occurs to at least one site outside the region involved in CpG recognition. No modulation in response to 5(me)C is observed with the bacterial enzyme M.SssI, which lacks the large N-terminal regulatory domain found in Dnmt1. We suggest that this allosteric modulation involves the N-terminal domain of Dnmt1. (C) 2003 Elsevier Inc. All rights reserved.

 

Flynn, J., et al. (2003). "A potent cell-active allosteric inhibitor of murine DNA cytosine C-5 methyltransferase." Journal of Biological Chemistry 278(10): 8238-8243.

                The major DNA cytosine methyltransferase isoform in mouse erythroleukemia cells, Dnmt1, exhibits potent dead-end inhibition with a single-stranded nucleic acid by binding to an allosteric site on the enzyme. The previously reported substrate inhibition with double-stranded substrates also involves binding to an allosteric site. Thus, both forms of inhibition involve ternary enzyme-DNA-DNA complexes. The inhibition potency of the single-stranded nucleic acid is determined by the sequence, length, and most appreciably the presence of a single 5-methyleytosine residue. A single-stranded phosphorothioate derivative inhibits DNA methylation activity in nuclear extracts. Mouse erythroleukemia cells treated with the phosphorothioate inhibitor show a significant decrease in global genomic methylation levels. Inhibitor treatment of human colon cancer cells causes demethylation of the p16 tumor suppressor gene and subsequent p16 re-expression. Allosteric inhibitors of mammalian DNA cytosine methyltransferases, representing a new class of molecules with potential therapeutic applications, may be used to elucidate novel epigenetic mechanisms that control development.

 

Hiller, D. A., et al. (2003). "Simultaneous DNA binding and bending by EcoRV endonuclease observed by real-time fluorescence." Biochemistry 42(49): 14375-14385.

                The complete catalytic cycle of EcoRV endonuclease has been observed by combining fluorescence anisotropy with fluorescence resonance energy transfer (FRET) measurements. Binding, bending, and cleavage of substrate oligonucleotides were monitored in real time by rhodamine-x anisotropy and by FRET between rhodamine and fluorescein dyes attached to opposite ends of a 14-mer DNA duplex. For the cognate GATATC site binding and bending are found to be nearly simultaneous, with association and bending rate constants of (1.45-1.6) x 10(8) M-1 s(-1). On the basis of the measurement of k(off) by a substrate-trapping approach, the equilibrium dissociation constant of the enzyme-DNA complex in the presence of inhibitory calcium ions was calculated as 3.7 x 10(-12) M from the kinetic constants. Further, the entire DNA cleavage reaction can be observed in the presence of catalytic Mg2+ ions. These measurements reveal that the binding and bending steps occur at equivalent rates in the presence of either Mg2+ or Ca2+, while a slow decrease in fluorescence intensity following bending corresponds to k(cat), which is limited by the cleavage and product dissociation steps. Measurement of k(on) and k(off) in the absence of divalent metals shows that the DNA binding affinity is decreased by 5000-fold to 1.4 x 10(-8) M, and no bending could be detected in this case. Together with crystallographic studies, these data suggest a model for the induced-fit conformational change in which the role of divalent metal ions is to stabilize the sharply bent DNA in an orientation suitable for accessing the catalytic transition state.

 

Lindstrom, W. M., et al. (2003). "Functional analysis of BamHI DNA cytosine-N-4 methyltransferase." Journal of Molecular Biology 325(4): 711-720.

                We show that the kinetic mechanism of the DNA (cytosine-N-4-)-methyltransferase M.BamHI, which modifies the underlined cytosine (GGAT (C) under barC), differs from cytosine C-5 methyltransferases, and is similar to that observed with adenine N-6 methyltransferases. This suggests that the obligate order of ternary complex assembly and disassembly depends on the type of methylation reaction. In contrast, the single-turnover rate of catalysis for M.Bam HI (0.10 s(-1)) is closer to the DNA (cytosine-C-5-)-methyltransferases (0.14 s(-1)) than the DNA (adenine-N-6-)-methyltransferases (>200 s(-1)). The nucleotide flipping transition dominates the singleturnover constant for adenine N-6 methyltransferases, and, since the disruption of the guanine-cytosine base-pair is essential for both types of cytosine DNA methyltransferases, this transition may be a common, rate-limiting step for methylation for these two enzyme subclasses. The similar overall rate of catalysis by M.Bam HI and other DNA methyltransferases is consistent with a common rate-limiting catalytic step of product dissociation. Our analyses of M.BamHI provide functional insights into the relationship between the three different classes of DNA methyltransferases that complement both prior structural and evolutionary insights. (C) 2003 Elsevier Science Ltd. All rights reserved.

 

Malygin, E. G., et al. (2003). "DNA (cytosine-N-4-)- and -(adenine-N-6-)-methyltransferases have different kinetic mechanisms but the same reaction route - A comparison of M.BamHI and T4 Dam." Journal of Biological Chemistry 278(18): 15713-15719.

                We studied the kinetics of methyl group transfer by the BamHI DNA- (cytosine-N-4-)-methyltransferase (MTase) from Bacillus amyloliquefaciens to a 20-mer oligodeoxynucleotide duplex containing the palindromic recognition site GGATCC. Under steady state conditions the BamHI MTase displayed a simple kinetic behavior toward the 20-mer duplex. There was no apparent substrate inhibition at concentrations much higher than the K. for either DNA (100-fold higher) or S-adenosyl-L-methionine (AdoMet) (20-fold higher); this indicates that dead-end complexes did not form in the course of the methylation reaction. The DNA methylation rate was analyzed as a function of both substrate and product concentrations. It was found to exhibit product inhibition patterns consistent with a steady state random bi-bi mechanism in which the dominant order of substrate binding and product release (methylated DNA, DNA(Me), and S-adenosyl-L-homocysteine, AdoHcy) was Ado-Met down arrow DNA down arrow DNA(Me) up arrow AdoHcy up arrow. The M.BamHI kinetic scheme was compared with that for the T4 Dam (adenine-N-6-)-MTase. The two differed with respect to an effector action of substrates and in the rate-limiting step of the reaction (product inhibition patterns are the same for the both MTases). From this we conclude that the common chemical step in the methylation reaction, methyl transfer from AdoMet to a free exocyclic amino group, is not sufficient to dictate a common kinetic scheme even though both MTases follow the same reaction route.

 

Malygin, E. G., et al. (2003). "Bacteriophage T4Dam (DNA-(adenine-N-6)-methyltransferase) - Evidence for two distinct stages of methylation under single turnover conditions." Journal of Biological Chemistry 278(43): 41749-41755.

                We compared the (pre) steady-state and single turnover methylation kinetics of bacteriophage T4Dam (DNA-(adenine-N-6)-methyltransferase)-mediated methyl group transfer from S-adenosyl-L-methionine (AdoMet) to oligodeoxynucleotide duplexes containing a single recognition site (palindrome 5'-GATC/5'-GATC) or some modified variant. T4Dam-AdoMet functions as a monomer under steady-state conditions (enzyme/DNA << 1), whereas under single turnover conditions (enzyme/DNA > 1), a catalytically active complex containing two Dam-AdoMet molecules is formed initially, and two methyl groups are transferred per duplex (to produce a methylated duplex and S-adenosyl-L-homocysteine (AdoHcy)). We propose that the single turnover reaction proceeds in two stages. First, two preformed T4Dam-AdoMet complexes bind opposite strands of the unmodified target site, and one enzyme molecule catalyzes the rapid transfer of the AdoMet-methyl group (k(meth1) = 0.21 s(-1)); this is 2.5-fold slower than the rate observed with monomeric T4Dam-AdoMet bound under pre-steady-state conditions for burst determination. In the second stage, methyl transfer to adenine in GATC on the complementary strand occurs at a rate that is 1 order of magnitude slower (k(meth2) = 0.023 s(-1)). We suggest that under single turnover conditions, methylation of the second strand is rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. The hemimethylated duplex 5'-GATC/5'-GMTC also interacts with T4Dam-AdoMet complexes in two stages under single turnover reaction conditions. The first stage (kmeth1) reflects methylation by dimeric T4Dam-AdoMet productively oriented to the strand with the adenine residue capable of methylation. The slower second stage (kmeth2) reflects methylation by enzyme molecules non-productively oriented to the GMTC chain, which then have to re-orient to the opposite productive chain. Substitutions of bases and deletions in the recognition site affect the kinetic parameters in different fashions. When the GAT portion of GATC was disrupted, the proportion of the initial productive enzyme-substrate complexes was sharply reduced.

 

Oza, J. and N. O. Reich (2003). "The role of DNA adenine methylation in Actinobacillus actinomycetem-comitans." Journal of Dental Research 82: B323-B323.

               

Svedruzic, Z. M. and N. O. Reich (2002). "Enzymatic properties of mouse cytosine DNA methyltransferase DNMT1." Abstracts of Papers of the American Chemical Society 223: C75-C75.

               

Yun, C. S., et al. (2002). "Enzymatic manipulation of DNA - Nanomaterial constructs." J Am Chem Soc 124(26): 7644-7645.

               

Eberhard, J., et al. (2001). "Cloning, sequence analysis and heterologous expression of the DNA adenine-(N-6) methyltransferase from the human pathogen Actinobacillus actinomycetemcomitans." Fems Microbiology Letters 195(2): 223-229.

                We cloned and sequenced the DNA adenine-N-6 methyltransferase gene of the human pathogen Actinobacillus actinomycetemcomitans (M.AacDAM). Restriction digestion shows that the enzyme methylates adenine in the sequence GATC. Expression of the enzyme in a DAM(-) background shows in vivo activity. A PSI-BLAST search revealed that M.AacDAM is most related to M.HindIV. M.EcoDAM, M.StyDAM. and M.Small. The ClustalW alignment shows highly conserved regions in the enzyme characteristic for type a MTases. Phylogenetic tree analysis shows a cluster of enzymes recognizing the sequence GATC, within a branch of orphan MTases harboring M.AacDAM, The cloning and sequencing of this first methyltransferase gene described for A. actinomycetemcomitans open the path for studies on the potential regulatory impact of DNA methylation on gene regulation and virulence in this organism. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

 

Malygin, E. G., et al. (2001). "DNA-(N4-cytosine)-methyltransferase from Bacillus amyloliquefaciens: Kinetic and substrate-binding properties." Molekulyarnaya Biologiya (Moscow) 35(1): 42-51.

                Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amyloliquefaciens (BamHI MTase, 49 kDa) with a 20-mer oligonucleotide duplex containing the palindrome recognition site GGATCC was studied by methods of steady-state and presteady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaric aldehyde. In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward a 20-mer oligonucleotide substrate. A linear dependence was observed for the reaction rate on the enzyme concentration and a Michaelis dependence of the reaction rate on the concentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl group donor, and DNA, the methyl group acceptor. In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme concentration being substantially lower then the concentrations of substrates. The kcat values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s-1. The Km values for the duplex and SAM are 0.35 and 1.6 muM, respectively. An analysis of single turnover kinetics (at limiting concentration of the 20-mer oligonucleotide duplex) revealed the following characteristics of the BamHI MTase-dependent methylation of DNA. The value of rate constant of the DNA metylation step at the enzyme saturating concentration is on average 0.085 s-1, which is only 1.6 times higher than the value determined in steady-state conditions. Only one of two target cytidine residues was methylated in the course of the enzyme single turnover, which coincides with the earlier data on EcoRI MTase. Regardless of the order of the enzyme preincubation with SAM and DNA, both curves for the single turnover methylation are comparable. These results are consistent with the model of the random order of the productive ternary enzyme-substrate complex formation. In contrast to the relatively simple kinetic behavior of BamHI MTase in the steady-state reaction are the data on the enzyme binding of DNA. In gel retardation experiments, there was no stoichiometrically simple complexes with the oligonucleotide duplex even at low enzyme concentrations. The molecular mass of the complexes was so high that they did not enter 12% PAG. In experiments on crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer. Introduction of substoichiometric amounts of DNA into the reaction mixture results in pronounced multimerization of the enzyme. However, addition of SAM in saturating concentration at an excess of the oligonucleotide duplex over BamHI MTase converts most of the enzyme into a monomeric state.

 

Malygin, E. G., et al. (2001). "DNA-(N4-cytosine)-Methyltransferase from Bacillus amyloliquefaciens: Kinetic and substrate-binding properties." Molecular Biology 35(1): 35-44.

                Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amyloliquefaciens (BamHI MTase, 49 kDa) with a 20-mer duplex containing a palindromic recognition site GGATCC was studied by methods of steady-state and pre-steady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaraldehyde. In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward the 20-mer substrate. A linear dependence was observed for the reaction rate on the enzyme concentration and a Michaelis dependence of the reaction rate on the concentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl group donor, and DNA, the methyl group acceptor. In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme concentration being substantially lower than the concentrations of substrates. The k(cat) values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s(-1). The K-M values for the duplex and SAM are 0.35 and 1.6 muM, respectively. An analysis of single turnover kinetics (at limiting concentration of the 20-mer duplex) revealed the following characteristics of the BamHI MTase-dependent methylation of DNA. The value of rate constant of the DNA methylation step at the enzyme saturating concentration is on average 0.085 s(-1), which is only 1.6 times higher than the value determined in steady-state conditions. Only one of two target cytidine residues was methylated in a single turnover of the enzyme, which coincides with the earlier data on EcoRI MTase. Regardless of the order of enzyme preincubation with SAM and DNA, both curves for the single turnover methylation are comparable. These results are consistent with the model of the random order of the productive ternary enzyme-substrate complex formation. In contrast to the relatively simple kinetic behavior of BamHI MTase in the steady-state reaction are the data on the enzyme binding with DNA. In gel retardation experiments, there was no stoichiometrically simple complex with the oligonucleotide duplex even at low enzyme concentrations. The molecular mass of the complexes was so high that they did not enter 12% PAG. In experiments on crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer. Introduction of substoichiometric amounts of DNA into the reaction mixture results in pronounced multimerization of the enzyme. However, addition of SAM in saturating concentration at an excess of the oligonucleotide duplex over BamHI MTase converts most of the enzyme into a monomeric state.

 

Malygin, E. G., et al. (2001). "A dual role for substrate S-adenosyl-L-methionine in the methylation reaction with bacteriophage T4 Dam DNA- N6-adenine -methyltransferase." Nucleic Acids Research 29(11): 2361-2369.

                The fluorescence of P-aminopurine ((2)A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase. With an unmethylated target ((2)A/A duplex) or its methylated derivative ((2)A/(m)A duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with (2)A being flipped out of the DNA helix. Though neither S-adenosyl-L-homocysteine nor sinefungin had any significant effect, addition of substrate S-adenosyl-L-methionine (AdoMet) sharply reduced the Dam-induced fluorescence with these complexes. In contrast, AdoMet had no effect on the fluorescence increase produced with an (2)A/(2)A double-substituted duplex. Since the (2)A/(m)A duplex cannot be methylated, the AdoMet-induced decrease in fluorescence cannot be due to methylation per se, We propose that T4 Dam alone randomly binds to the asymmetric (2)A/A and (2)A/(m)A duplexes, and that AdoMet induces an allosteric T4 Dam conformational change that promotes reorientation of the enzyme to the strand containing the native base. Thus, AdoMet increases enzyme binding-specificity, in addition to serving as the methyl donor, The results of pre-steady-state methylation kinetics are consistent with this model.

 

Brunhuber, N. M. W., et al. (2000). "Steady-state kinetic mechanism of recombinant avocado ACC oxidase: Initial velocity and inhibitor studies." Biochemistry 39(35): 10730-10738.

                The gaseous plant hormone ethylene modulates a wide range of biological processes, including fruit ripening. It is synthesized by the ascorbate-dependent oxidation of 1-aminocyclopropyl-1-carboxylate (ACC), a reaction catalyzed by ACC oxidase. Recombinant avocado (Persea americana) ACC oxidase was expressed in Esherichia coli and purified in milligram quantities, resulting in high levels of ACC oxidase protein and enzyme activity. An optimized assay for the purified enzyme was developed that takes into account the inherent complexities of the assay system. Fe(II) and ascorbic acid form a binary complex that is not the true substrate for the reaction and enhances the degree of ascorbic acid substrate inhibition. The K-d value for Fe(II) (40 nM, free species) and the K-m's for ascorbic acid (2.1 mM), ACC (62 mu M), and O-2 (4 mu M) were determined. Fe(II) and ACC exhibit substrate inhibition, and a second metal binding site is suggested. Initial velocity measurements and inhibitor studies were used to resolve the kinetic mechanism through the final substrate binding step. Fe(II) binding is followed by either ascorbate or ACC binding, with ascorbate being preferred. This is followed by the ordered addition of molecular oxygen and the last substrate, leading to the formation of the catalytically competent complex. Both Fe(II) and O-2 are in thermodynamic equilibrium with their enzyme forms. The binding of a second molecule of ascorbic acid or ACC leads to significant substrate inhibition. BCC and ascorbate analogues were used to confirm the kinetic mechanism and to identify important determinants of substrate binding.

 

Lindstrom, W. M., et al. (2000). "Reconciling structure and function in HhaI DNA cytosine-C-5 methyltransferase." Journal of Biological Chemistry 275(7): 4912-4919.

                Pre-steady state partitioning analysis of the HhaI DNA methyltransferase directly demonstrates the catalytic competence of the enzyme DNA complex and the lack of catalytic competence of the enzyme-S-adenosyl-L-methionine (AdoMet) complex. The enzyme.AdoMet complex does form, albeit with a 50-fold decrease in affinity compared with the ternary enzyme.AdoMet.DNA complex. These findings reconcile the distinct binding orientations previously observed within the binary enzyme.AdoMet and ternary enzyme.S-adenosyl-L-homocysteine.DNA crystal structures. The affinity of the enzyme for DNA is increased 900-fold in the presence of its cofactor, and the preference for hemimethylated DNA is increased to 12-fold over unmethylated DNA We suggest that this preference is partially due to the energetic cost of retaining a cavity in place of the B-methyl moiety in the ternary complex with the unmethylated DNA, as revealed by the corresponding crystal structures. The hemi- and unmethylated substrates alter the fates and lifetimes of discrete enzyme substrate intermediates during the catalytic cycle. Hemimethylated substrates partition toward product formation versus dissociation significantly more than unmethylated substrates. The mammalian DNA cytosine-C-5 methyltransferase Dnmt1 shows an even more pronounced partitioning toward product formation.

 

Malygin, E. G., et al. (2000). "Pre-steady state kinetics of bacteriophage T4 Dam DNA- N-6-adenine methyltransferase: interaction with native (GATC) or modified sites." Nucleic Acids Research 28(21): 4207-4211.

                The DNA methyltransferase of bacteriophage T4 (T4 Dam MTase) recognizes the palindromic sequence GATC, and catalyzes transfer of the methyl group from S-adenosyl-L-methionine (AdoMet) to the N-6-position of adenine [generating N-6-methyladenine and S-adenosyl-L-homocysteine (AdoHcy)]. Pre-steady state kinetic analysis revealed that the methylation rate constant k(meth) for unmethylated and hemimethylated substrates (0.56 and 0.47 s(-1), respectively) was at least 20-fold larger than the overall reaction rate constant k(cat) (0.023 s(-1)). This indicates that the release of products is the rate-limiting step in the reaction. Destabilization of the target-base pair did not alter the methylation rate, indicating that the rate of target nucleoside flipping does not limit k(meth). Preformed T4 Dam MTase-DNA complexes are less efficient than preformed T4 Dam MTase-AdoMet complexes in the first round of catalysis. Thus, this data is consistent with a preferred route of reaction for T4 Dam MTase in which AdoMet is bound first; this preferred reaction route is not observed with the DNA-[C5-cytosine]-MTases.

 

Martin, A. M., et al. (2000). "Simultaneous real time measurement of binding, bending and product release by the EcoRV endonuclease." Biophysical journal 78(1): 395A-395A.

               

Szegedi, S. S., et al. (2000). "Substrate binding in vitro and kinetics of RsrI N6-adenine DNA methyltransferase." Nucleic Acids Research 28(20): 3962-3971.

                Rsrl [N6-adenine] DNA methyltransferase (M . Rsrl), which recognizes GAATTC and is a member of a restriction-modification system in Rhodobacter sphaeroides, was purified to >95% homogeneity using a simplified procedure involving two ion exchange chromatographic steps. Electrophoretic gel retardation assays with purified M . Rsrl were performed on unmethylated, hemimethylated, dimethylated or non-specific target DNA duplexes (25 bp) in the presence of sinefungin, a potent inhibitory analog of AdoMet, M . Rsrl binding was affected by the methylation status of the DNA substrate and was enhanced by the presence of the cofactor analog. M . Rsrl bound DNA substrates in the presence of sinefungin with decreasing affinities: hemimethylated > unmethylated > dimethytated >> non-specific DNA, Gel retardation studies with DNA substrates containing an abasic site substituted for the target adenine DNA provided evidence consistent with M . Rsrl extruding the target base from the duplex. Consistent with such base flipping, an similar to1.7-fold fluorescence intensity increase was observed upon stoichiometric addition of M . Rsrl to hemimethylated DNA containing the fluorescent analog P-aminopurine in place of the target adenine, Pre-steady-state kinetic and isotope-partitioning experiments revealed that the enzyme displays burst kinetics, confirmed the catalytic competence of the M . Rsrl-AdoMet complex and eliminated the possibility of an ordered mechanism where DNA is required to bind first. The equilibrium dissociation constants for AdoMet, AdoHcy and sinefungin were determined using an intrinsic tryptophan fluorescence-quenching assay.

 

Wong, D. L. and N. O. Reich (2000). "Identification of tyrosine 204 as the photo-cross-linking site in the DNA - EcoRI DNA methyltransferase complex by electrospray ionization mass spectrometry." Biochemistry 39(50): 15410-15417.

                We describe a highly sensitive strategy combining laser-induced photo-cross-linking and HPLC-based electrospray ionization mass spectrometry to identify amino acid residues involved in protein-DNA recognition. The photoactivatible cross-linking thymine isostere, 5-iodoracil, was incorporated at a single site within the sequence recognized by EcoRI DNA methyltransferase (GAATTC). UV irradiation of the DNA-protein complex at 313 nm results in a >60% cross-linking yield. SDS-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the covalent cross-linked complex. The total mass is consistent with covalent bond formation between one strand of DNA and the protein with 1:1 stoichiometry. Protease digestion of the cross-linked complex yields several peptide-DNA adducts that were purified by anion-exchange column chromatography. A combination of mass spectrometric analysis and amino acid sequencing revealed that tyrosine 204 was cross-linked to the DNA. Electrospray mass spectrometric analysis of the peptide-nucleoside adduct confirmed this assignment. Tyrosine 204 resides in a peptide motif previously thought to be involved in AdoMet binding and methyl transfer. Thus, amino acids within loop segments but outside of "DNA binding" motifs can be critical to DNA recognition. Our method provides an accurate characterization of picomole quantities of DNA-protein complexes.