Selected Publications
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- The Role of High Affinity Non-specific DNA Binding by Lrp in Transcriptional Regulation and DNA Organization
Stacey N. Peterson, Frederick W. Dahlquist, and Norbert O. Reich. J. Mol. Bio. 2007 June; 369 (5), 1307-1317.Transcriptional regulatory proteins typically bind specific DNA sequences with not, vert, similar103–107-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 not, vert, similar20–400-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 not, vert, similar20-fold decrease in binding affinity from specific DNA is dependent upon cryptic binding sites, including the sequence GN2-3TTT and A-tracts. Removal of these sites still results in high binding affinity, only not, vert, similar70-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 non-specific DNA binding. When considering this cooperativity of Lrp binding, the binding selectivity to specific sites may increase to a maximum of not, vert, similar400-fold. Neither leucine nor the pap-specific local regulator PapI alter Lrp's non-specific 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.
[pdf] - Chemically Patterned Microspheres for Controlled Nanoparticle Assembly in the Construction of SERS Hot Spots
Gary Braun, Ioana Pavel, Andrew R. Morrill, Dwight S. Seferos, Guillermo C. Bazan, Norbert O. Reich, and Martin Moskovits. J. Am. Chem. Soc. 2007 June; 129 (25), 7760 -7761.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., under 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.
[pdf] - Long-Range Structural and Dynamical Changes Induced by Cofactor Binding in DNA Methyltransferase M.HhaI
Hongjun Zhou, Whitney Shatz, Matthew M. Purdy, Nick Fera, Frederick W. Dahlquist, and Norbert O. Reich. Biochemistry 2007 May;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 (GCGC) 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 Å 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.
[pdf] - A Heterogeneous PNA-Based SERS Method for DNA Detection
Laura Fabris, Mark Dante, Gary Braun, Seung Joon Lee, Norbert O. Reich, Martin Moskovits, Thuc-Quyen Nguyen, and Guillermo C. Bazan. J. Am. Chem. Soc. 2007 Apr; 129(19), 6086-6087.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.
[pdf] - Inactivation of DNA adenine methyltransferase alters virulence factors in Actinobacillus actinomycetemcomitans
NH. Wu, J. E. Lippmann, J. P. Oza, M. Zeng, P. Fives-Taylor, N. O. Reich. Oral Microbio. and Immun. 2006 Aug; 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.
[pdf] - Selective Inhibitors of Bacterial DNA Adenine Methyltransferases
NEDA MASHHOON, CYNTHIA PRUSS, MICHAEL CARROLL, PAUL H. JOHNSON, and NORBERT O. REICH. J. Biomol. Screen. 2006 June;11:497.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 smallmolecule DNA methyltransferase inhibitors provide useful reagents to probe the role of DNA methylation and may form the basis of developing novel antibiotics. (Journal of Biomolecular Screening 2006:1-14)
[pdf] - Conformational Transitions as Determinants of Specificity for the DNA Methyltransferase EcoRI
Ben Youngblood and Norbert O. Reich. J. Biol. Chem. 2006 Sept;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 ~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.
[pdf] - Determinants of Sequence-Specific DNA Methylation: Target Recognition and Catalysis Are Coupled in M.HhaI
Ben Youngblood, Fabian Buller, and Norbert O. Reich. Biochemistry. 2006 Dec;45(51):15563 -15572.Sequence specificity studies of the wild-type bacterial DNA cytosine C5 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 kcat/KDDNA is decreased 9000-80000-fold relative to the cognate site, manifested through changes in methylation, or a prior step, and changes in KDDNA. 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.
[pdf] - The Role of Arg165 Towards Base Flipping, Base Stabilization and Catalysis in M.HhaI
Fa-Kuen Shieh, Ben Youngblood and Norbert O. Reich. J Mol Biol. 2006 Sept;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 not, vert, similar16 and 190-fold respectively, thus confirming the importance of this motif. However, these kinetic changes cannot account for the mutant's observed 105-fold decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure (2.79 Å resolution) shows the target cytosine to be positioned not, vert, similar30° into the major groove, which is consistent with a major groove pathway for nucleotide flipping. The pyrimidine-sugar χ angle is rotated to approximately +171°, from a range of −95° to −120° in B DNA, and −77° 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 Å resolution cocrystal structure of the WT M.HhaI/DNA complex.
We determined a high resolution structure of the WT M.HhaI/DNA complex (1.96 Å) 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.
[pdf] - Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI
Ben Youngblood, Fa-Kuen Shieh, Stephanie De Los Rios, John J. Perona, and Norbert O. Reich. J Mol Biol. 2006 Sept;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.
[pdf] - GATC Flanking Sequences Regulate Dam Activity: Evidence for how Dam Specificity may Influence pap Expression.
Peterson SN, Reich NO. J Mol Biol. 2006 Jan 20;355(3):459-72.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.
[pdf] - DNA methylation modulates Salmonella enterica serovar Typhimurium virulence in Caenorhabditis elegans
Javin P. Oza, Jimmy B. Yeh, Norbert O. Reich. FEMS Microbiology Letters 245 (2005) 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 dam-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.
[pdf] - Controlled Spacing of Cationic Gold Nanoparticles by Nanocrown RNA
Alexey Y. Koyfman, Gary Braun, Sergei Magonov, Arkadiusz Chworos, Norbert O. Reich, and Luc Jaeger. J. AM. CHEM. SOC. 2005, 127, 11886-11887.JACS Communication
[pdf] - In This Issue of PNAS: Analyzing enzyme dynamics
PNAS 2005 vol. 102 no. 4 957.PNAS Commentary
[pdf] - Nanometal Surface Energy Transfer in Optical Rulers, Breaking the FRET Barrier
C. S. Yun, A. Javier, T. Jennings, M. Fisher, S. Hira, S. Peterson, B. Hopkins, N. O. Reich, and G. F. Strouse. J. AM. CHEM. SOC. 2005, 127, 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 U 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 u) and follows a 1/R4 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 u). 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.
[pdf] - Gold Nanoparticle Decoration of DNA on Silicon
Gary Braun, Katsuhiko Inagaki, R. August Estabrook, D. K. Wood, Eran Levy, A. N. Cleland, Geoffrey F. Strouse, and Norbert O. Reich. Langmuir 2005, 21, 10699-10701.Electrostatic assembly of cationic nanoparticles onto the negatively charged backbone of double-stranded DNAhas 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 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.
[pdf] - DNA Cytosine C5 Methyltransferase Dnmt1: Catalysis-Dependent Release of Allosteric Inhibition�
Zeljko M. Svedruzic and Norbert O. Reich. Biochemistry 2005, 44, 9472-9485.We followed the cytosine C5 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 cysteinecytosine 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 limination 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.
[pdf] - Statistical coevolution analysis and molecular dynamics: Identification of amino acid pairs essential for catalysis
R. August Estabrook, Jia Luo, Matthew M. Purdy, Vyas Sharma, Paul Weakliem, Thomas C. Bruice, and Norbert O. Reich. PNAS 2005 vol. 102 no. 4 994-999.Molecular dynamics (MD) simulations of HhaI 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 (SCAMD) are colocalized and form stabilizing interactions; the anticorrelated pairs are separated on average by 30 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 SCAMD approach.
[pdf] - The Mechanism of Target Base Attack in DNA Cytosine Carbon 5 Methylation
Zeljko M. Svedruzic and Norbert O. Reich. Biochemistry 2004, 43, 11460-11473.We measured the tritium exchange reaction on cytosine C5 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 presteady- 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 Gln237Trp mutant or in the cytosine C5 exchange reaction in the absence of the cofactor.
[pdf] - Functional Characterization of Escherichia coli DNA Adenine Methyltransferase, a Novel Target for Antibiotics
Neda Mashhoon, Michael Carroll, Cynthia Pruss, Joerg Eberhard, Sawako Ishikawa, R. August Estabrook, and Norbert Reich. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 50, Issue of December 10, pp. 52075�52081, 2004.We have characterized Escherichia coli DNA adenine methyltransferase, a critical regulator of bacterial virulence. Steady-state kinetics, product inhibition, and isotope exchange studies are consistent with a kinetic mechanism in which the cofactor S-adenosylmethionine binds first, followed by sequence-specific DNA binding and catalysis. The enzyme has a fast methyl transfer step followed by slower product release steps, and we directly demonstrate the competence of the enzyme cofactor complex. Methylation of adjacent GATC sites is distributive with DNA derived from a genetic element that controls the transcription of the adjacent genes. This indicates that the first methylation event is followed by enzyme release. The affinity of the enzyme for both DNA and S-adenosylmethionine was determined. Our studies provide a basis for further structural and functional analysis of this important enzyme and for the identification of inhibitors for potential therapeutic applications.
[pdf] - Novel inhibitors of bacterial DNA adenine methyltransferases: a new class of antibiotics
Neda Mashhoon, Cynthia Pruss, Michael Carroll, Paul H. Johnson, and Norbert O. Reich.We 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 FRET-based assay and recombinant DAM and CcrM, we screened a diverse chemical library. We identified five major structural classes of inhibitors comprised of over 350 compounds with molecular weights less than 500 and ClogP values less than 5: cyclopentaquinolines, phenyl vinyl furans, pyrimidinediones, 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, while 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 form the basis of developing an entirely novel class of antibiotics.
[pdf] - The Coupling of Tight DNA Binding and Base Flipping, Identification of a Conserved Structural Motif in Base Flipping Enzymes
R. August Estabrook, Rebecca Lipson, Ben Hopkins, and Norbert Reich. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 30, Issue of July 23, pp. 31419-31428, 2004.Val121 is positioned immediately above the extrahelical cytosine in HhaI DNA C5-cytosine methyltransferase, and replacement with alanine dramatically interferes with base flipping and catalysis. DNA binding and kcat are decreased 105-fold for the Val121 3 Ala mutant that has a normal circular dichroism spectrum and AdoMet affinity. The magnitude of this loss of function is comparable with removal of the essential catalytic Cys81. Surprisingly, DNA binding is completely recovered (increase of 105-fold) with a DNA substrate lacking the target cytosine base (abasic). Thus, interfering with the base flipping transition results in a dramatic loss of binding energy. Our data support an induced fit mechanism in which tight DNA binding is coupled to both base flipping and protein loop rearrangement. The importance of the proximal protein segment (His121-Thr132) in maintaining this critical interaction between Val121 and the flipped cytosine was probed with single site alanine substitutions. None of these mutants are significantly altered in secondary structure, AdoMet or DNA affinity, kmethylation, kinactivation, or kcat. Although Val121 plays a critical role in both extrahelical base stabilization and catalysis, its position and mobility are not influenced by individual residues in the adjacent peptide region. Structural comparisons with other DNA methyltransferases and DNA repair enzymes that stabilize extrahelical nucleotides reveal a motif that includes a positively charged or polar side chain and a hydrophobic residue positioned adjacent to the target DNA base and either the 5- or 3-phosphate.
[pdf] - Simultaneous DNA Binding, Bending, and Base Flipping: Evidence for a Novel M.EcoRI Methyltransferase-DNA Complex
Ben B. Hopkins and Norbert O. Reich. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 35, Issue of August 27, pp. 37049�37060, 2004.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.
[pdf] - A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes
Richard J. Roberts, Marlene Belfort, Shuang-yong Xu, Norbert Reich, et al. Nucleic Acids Research, 2003, Vol. 31, No. 7 1805-1812.A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.
[pdf] - Murine DNA cytosine C5-methyltransferase: in vitro studies of de novo methylation spreading
Brandon E. Aubol and Norbert O. Reich. Biochemical and Biophysical Research Communications 310 (2003) 211?216.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 (5meC). 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 5meC moiety be attached to the DNA strand containing the CpG methylation target. Our results support a model in which 5meC binding by the enzyme occurs to at least one site outside the region involved in CpG recognition. No modulation in response to 5meC 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.
[pdf] - DNA (Cytosine-N4-)- and -(Adenine-N6-)-methyltransferases Have Different Kinetic Mechanisms but the Same Reaction Route
Ernst G. Malygin, Victor V. Zinoviev, Alexey A. Evdokimov, William M. Lindstrom, Jr., Norbert. O. Reich, and Stanley Hattman. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 18, Issue of May 2, pp. 15713?15719, 2003.We studied the kinetics of methyl group transfer by the BamHI DNA-(cytosine-N4-)-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 Km for either DNA (100-fold higher) or S-adenosyl-Lmethionine (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, DNAMe, and S-adenosyl-L-homocysteine, AdoHcy) was Ado- Met2DNA2DNAMe1AdoHcy1. The M.BamHI kinetic scheme was compared with that for the T4 Dam (adenine- N6-)-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.
[pdf] - Functional Analysis of BamHI DNA Cytosine-N4 Methyltransferase
William M. Lindstrom Jr, Ernst G. Malygin, Lidiya G. Ovechkina, Victor V. Zinoviev, and Norbert O. Reich. J. Mol. Biol. (2003) 325, 711-720.We show that the kinetic mechanism of the DNA (cytosine-N4-)-methyltransferase M.BamHI, which modifies the underlined cytosine GATCC differs from cytosine C5 methyltransferases, and is similar to that observed with adenine N6 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.BamHI (0.10 s21) is closer to the DNA (cytosine-C5-)-methyltransferases (0.14 s21) than the DNA (adenine-N6-)-methyltransferases (.200 s21). The nucleotide flipping transition dominates the singleturnover constant for adenine N6 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, ratelimiting 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.Bam HI provide functional insights into the relationship between the three different classes of DNA methyltransferases that complement both prior structural and evolutionary insights.
[pdf] - A Potent Cell-active Allosteric Inhibitor of Murine DNA Cytosine C5 Methyltransferase
James Flynn, Jing-Yuan Fang, Judy A. Mikovits, and Norbert O. Reich. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 10, Issue of March 7, pp. 8238�8243, 2003.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 doublestranded 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-methylcytosine 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.
[pdf] - Simultaneous DNA Binding and Bending by EcoRV Endonuclease Observed by Real-Time Fluorescence
David A. Hiller, Jonathan M. Fogg, Amy M. Martin, Joseph M. Beechem, Norbert O. Reich, and John J. Perona. Biochemistry 2003, 42, 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 koff 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 kcat, which is limited by the cleavage and product dissociation steps. Measurement of kon and koff 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.
[pdf] - Enzymatic Manipulation of DNA-Nanomaterial Constructs
C. Steven Yun, Gregory A. Khitrov, Danielle E. Vergona, Norbert O. Reich, and Geoffrey F. Strouse. J. AM. CHEM. SOC. 2002, 124, 7644-7645.JACS Communication
[pdf] - Cloning, sequence analysis and heterologous expression of the DNA adenine-(N6) methyltransferase from the human pathogen Actinobacillus actinomycetemcomitans
Jorg Eberhard, Javin Oza, Norbert O. Reich. FEMS Microbiology Letters 195 (2001) 223-229.We cloned and sequenced the DNA adenine-N6 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 DAM3 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.SmaII. 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.
[pdf] - Reconciling Structure and Function in HhaI DNA Cytosine-C-5 Methyltransferase
William M. Lindstrom, Jr., James Flynn, and Norbert O. Reich. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 7, Issue of February 18, pp. 4912?4919, 2000.Pre-steady state partitioning analysis of the HhaI DNA methyltransferase directly demonstrates the catalytic competence of the enzymezDNA complex and the lack of catalytic competence of the enzymezS-adenosyl- L-methionine (AdoMet) complex. The enzymezAdoMet complex does form, albeit with a 50-fold decrease in affinity compared with the ternary enzymezAdoMetzDNA complex. These findings reconcile the distinct binding orientations previously observed within the binary enzymezAdoMet and ternary enzymezS-adenosyl-LhomocysteinezDNA 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 5-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 enzymezsubstrate 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.
[pdf] - Identification of Tyrosine 204 as the Photo-Cross-Linking Site in the DNA-EcoRI DNA Methyltransferase Complex by Electrospray Ionization Mass Spectrometry
David L. Wong and Norbert O. Reich. Biochemistry 2000, 39, 15410-15417.We describe a highly sensitive strategy combining laser-induced photo-cross-linking and HPLCbased 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.
[pdf] - DNA Bending by EcoRI DNA Methyltransferase Accelerates Base Flipping but Compromises Specificity
Barrett W. Allan, Ricardo Garcia, Karen Maegley, Jessica Mort, David Wong, William Lindstrom, Joseph M. Beechem, and Norbert O. Reich. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 27, Issue of July 2, pp. 19269-19275, 1999.EcoRI DNA methyltransferase was previously shown to bend its cognate DNA sequence by 52 ?and stabilize the target adenine in an extrahelical orientation. We describe the characterization of an EcoRI DNA methyltransferase mutant in which histidine 235 was selectively replaced with asparagine. Steady-state kinetic and thermodynamic parameters for the H235N mutant revealed only minor functional consequences: DNA binding affinity (KD DNA) was reduced 10-fold, and kcat was decreased 30%. However, in direct contrast to the wild type enzyme, DNA bending within the mutant enzyme- DNA complexes was not observed by scanning force microscopy. The bending-deficient mutant showed enhanced discrimination against the methylation at nontarget sequence DNA. This enhancement of enzyme discrimination was accompanied by a change in the rate-limiting catalytic step. No presteady-state burst of product formation was observed, indicating that the chemistry step (or prior event) had become rate-limiting for methylation. Direct observation of the base flipping transition showed that the lack of burst kinetics was entirely due to slower base flipping. The combined data show that DNA bending contributes to the correct assembly of the enzyme-DNA complex to accelerate base flipping and that slowing the rate of this precatalytic isomerization can enhance specificity.
[pdf] - Measurement of the Absolute Temporal Coupling between DNA Binding and Base Flipping
Barrett W. Allan, Norbert O. Reich, and Joseph M. Beechem. Biochemistry 1999, 38, 5308-5314.The absolute temporal couplings between DNA binding and base flipping were examined for the EcoRI DNA methyltransferase. The binding event (monitored using rhodamine-x fluorescence anisotropy) was monophasic with a second-order on-rate of 1.1x10^7 M-1 s-1. Base-flipping kinetics (monitored using 2-aminopurine fluorescence intensity) were essentially synchronous with the binding kinetics, with less than a 4 ms delay between enzyme binding and target base flipping. The 4 ms delay translates into a base-flipping rate of at least 195 s-1, when the data are analyzed in terms of a sequential DNA binding and base-flipping reaction mechanism. Synchrony of binding and base flipping was only observed during the first 80 of the reaction, and an additional 20 base-flipping signal occurred well after DNA binding was complete. This additional 2AP fluorescence change, with an effective rate of 0.55 s-1, is an intramolecular isomerization reaction which greatly accelerates the dissociation of the enzyme from DNA. The correlation between the dissociation of the enzyme-DNA complex and the restacking of the extrahelical base also revealed a very tight coupling of these two events. Both dissociation and base restacking were found to be biphasic. These data are consistent with the following mechanism. The initial binding rate and base-flipping rates map very closely with previously determined pre-steady-state burst-rate kinetics for methyl transfer. Hence, binding, flipping, and methylation appear to occur in nearly a single concerted step. The bound complex then slowly isomerizes (0.1 s-1) to a distinct configuration that accelerates the product-release phase of the reaction.
The product-release enzyme configuration dissociates from DNA approximately 8 times faster than the initial bound complex (0.18 s-1 vs 0.024 s-1 ). When the enzyme dissociates from the DNA along the product-release pathway, the target base remains in an extrahelical conformation and restacks at a rate of only 0.6 s-1. This multicolor fluorescence kinetic approach directly measures the absolute temporal correlation between DNA binding and base flipping, with millisecond timing resolution. The data reveal that even when the B-DNA structure is altered in a radical manner (e.g., via base flipping), enzymes can perform this operation in a highly efficient, if not completely concerted manner.
[pdf] - Murine DNA (Cytosine-5-)-methyltransferase: Steady-State and Substrate Trapping Analyses of the Kinetic Mechanism
James Flynn and Norbert Reich. Biochemistry 1998, 37, 15162-15169.DNA (cytosine-5-)-methyltransferase is essential for viable mammalian development and has a central function in the determination and maintenance of epigenetic methylation patterns. Steady-state and substrate trapping studies were performed to better understand how the enzyme functions. The catalytic efficiency was dependent on substrate DNA length. A 14-fold increase in Km DNA was observed as the length decreased from 5000 to 100 base pairs and kcat decreased by a third. Steady-state analyses were used to identify the order of substrate addition onto the enzyme and the order of product release. Doublereciprocal patterns of velocity versus substrate concentration intersected far from the origin and were nearly parallel. The kinetic mechanism does not appear to change when the DNA substrate is either 6250 or 100 base pairs in length. Isotope trapping studies showed that the initial enzyme-AdoMet complex was not catalytically competent; however, the initial enzyme-poly(dIC-dIC) complex was observed to be competent for catalysis. Product inhibition studies also support a sequential ordered bi-bi kinetic mechanism in which DNA binds to the enzyme first, followed by S-adenosyl-L-methionine, and then the products S-adenosyl-L-homocysteine and methylated DNA are released. The proposed mechanism is similar to the mechanism proposed for M.HhaI, a bacterial DNA (cytosine-5-)-methyltransferase. Evidence for an enzyme-DNA-DNA ternary complex is also presented.
[pdf] - Direct Real Time Observation of Base Flipping by the EcoRI DNA Methyltransferase
Barrett W. Allan, Joseph M. Beechem, William M. Lindstrom, and Norbert O. Reich. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 4, Issue of January 23, pp. 2368-2373, 1998.DNA methyltransferases are excellent prototypes for investigating DNA distortion and enzyme specificity because catalysis requires the extrahelical stabilization of the target base within the enzyme active site. The energetics and kinetics of base flipping by the EcoRI DNA methyltransferase were investigated by two methods. First, equilibrium dissociation constants (KD DNA) were determined for the binding of the methyltransferase to DNA containing abasic sites or base analogs incorporated at the target base. Consistent with a base flipping mechanism, tighter binding to oligonucleotides containing destabilized target base pairs was observed. Second, total intensity stopped flow fluorescence measurements of DNA containing 2-aminopurine allowed presteadystate real time observation of the base flipping transition. Following the rapid formation of an enzyme-DNA collision complex, a biphasic increase in total intensity was observed. The fast phase dominated the total intensity increase with a rate nearly identical to kmethylation determined by rapid chemical quench-flow techniques (Reich, N. O., and Mashoon, N. (1993) J. Biol. Chem. 268, 9191-9193). The restacking of the extrahelical base also revealed biphasic kinetics with the recovered amplitudes from these off-rate experiments matching very closely to those observed during the base unstacking process. These results provide the first direct and continuous observation of base flipping and show that at least two distinct conformational transitions occurred at the flipped base subsequent to complex formation. Furthermore, our results suggest that the commitment to catalysis during the methylation of the target site is not determined at the level of the chemistry step but rather is mediated by prior intramolecular isomerization within the enzyme-DNA complex.
[pdf] - Electrospray ionization mass spectrometric characterization of photocrosslinked DNA-EcoRI DNA methyltransferase complexes
David L. Wong, James G. Pavlovich and Norbert O. Reich. Nucleic Acids Research, 1998, Vol. 26, No. 2 645-649.We describe a novel strategy combining photocrosslinking and HPLC-based electrospray ionization mass spectrometry to identify UV crosslinked DNA?rotein complexes. EcoRI DNA methyltransferase modifies the second adenine within the recognition sequence GAATTC. Substitution of 5-iodouracil for the thymine adjacent to the target base (GAATTC) does not detectably alter the DNA?rotein complex. Irradiation of the 5-iodouracil-substituted DNA?rotein complex at various wavelengths was optimized, with a crosslinking yield 60% at 313 nm after 1 min. No protein degradation was observed under these conditions. The crosslinked DNA?rotein complex was further analyzed by electrospray ionization mass spectrometry. The total mass is consistent with irradiation-dependent covalent bond formation between one strand of DNA and the protein. These preliminary results support the possibility of identifying picomole quantities of crosslinked peptides by similar strategies.
[pdf] - Peptide Mapping of the Murine DNA Methyltransferase Reveals a Major Phosphorylation Site and the Start of Translation
J. Fraser Glickman, James G. Pavlovich, and Norbert O. Reich. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 28, Issue of July 11, pp. 17851-17857, 1997.The murine DNA methyltransferase catalyzes the transfer of methyl groups from S-adenosylmethionine to cytosines within d(CpG) dinucleotides. The enzyme is necessary for normal embryonic development and is implicated in a number of important processes, including the control of gene expression and cancer. Metabolic labeling and high pressure liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) were performed on DNA methyltransferase purified from murine erythroleukemia cells. Serine 514 was identified as a major phosphorylation site that lies in a domain required for targeting of the enzyme to the replication foci. These results present a potential mechanism for the regulation of DNA methylation. HPLC-ESI-MS peptide mapping data demonstrated that the purified murine DNA methyltransferase protein contains the N-terminal regions predicted by the recently revised 5* gene sequences (Yoder, J. A., Yen, R.-W. C., Vertino, P. M., Bestor, T. H., and Baylin, S. B. (1996) J. Biol. Chem. 271, 31092-31097). The evidence suggests a start of translation at the first predicted methionine, with no alternate translational start sites. Our peptide mapping results provide a more detailed structural characterization of the DNA methyltransferase that will facilitate future structure/function studies.
[pdf] - Contribution of Facilitated Diffusion and Processive Catalysis to Enzyme Efficiency: Implications for the EcoRI Restriction-Modification System
Mark A. Surby and Norbert O. Reich. Biochemistry 1996, 35, 2201-2208.The contribution of nonspecific DNA to enzyme efficiency (kcat/Km) is described for a sequencespecific DNA-modifying enzyme. Our investigation focuses on the EcoRI DNA methyltransferase which transfers a methyl group from the cofactor S-adenosylmethionine to the second adenine in the doublestranded DNA sequence GAATTC. kcat/Km increases 4-fold as DNA length increases from 14 to 429 base pairs and increases 2-fold as the distance from the site to the nearest end is increased from 29 to 378 base pairs. No changes in kcat/Km result from further increases in either case. A facilitated diffusion mechanism is proposed in which the methyltransferase scans an average of 400 base pairs prior to dissociation from a DNA molecule. The methyltransferase was found to methylate two sites on a single DNA molecule in a distributive rather than a processive manner, suggesting that the enzyme dissociates from the DNA prior to release of the reaction product S-adenosylhomocysteine. A direct competition experiment with the EcoRI endonuclease shows the methyltransferase to be slightly more efficient at specific site location and catalysis. A rationale for the role of facilitated diffusion in this type II restrictionmodification system is proposed.
[pdf] - Facilitated Diffusion of the EcoRI DNA Methyltransferase Is Described by a Novel Mechanism
Mark A. Surby and Norbert O. Reich. Biochemistry 1996, 35, 2209-2217.The contribution of nonspecific DNA to binding parameters (Kd, koff, and kon) was determined for the EcoRI DNA methyltransferase under noncatalytic conditions. An increase in DNA size from 14 to 775 base pairs causes a 20-fold decrease in Kd, while koff remains constant over the same range. The calculated kon increases with longer substrates, consistent with a facilitated diffusion mechanism. However, the combined results deviate from the model developed to describe facilitated diffusion [Berg, O.G.,Winter, R. B., and von Hippel, P. H. (1981) Biochemistry 20, 6929-6948]. Our results were successfully simulated using numerical integration of a kinetic scheme invoking protein dissociation via the ends of DNA. Consistent with this scheme, the methyltransferase dissociates more slowly from a circularized DNA molecule than from the identical linearized form. The simulation strategy correctly models our data with the methyltransferase and should be generally useful for routine modeling of facilitated diffusion involving protein-DNA systems.
[pdf] - Targeted Base Stacking Disruption by the EcoRI DNA Methyltransferase
Barrett W. Allan and Norbert O. Reich. Biochemistry 1996, 35, 14757-14762.We describe a novel fluorescence-based assay for detecting DNA conformational alterations within enzyme-DNA complexes. The target adenine for EcoRI DNA methyltransferase (GAATTC) was replaced with 2-aminopurine, which fluoresces upon excitation at 310 nm. Addition of the methyltransferase to the duplex binding site results in a 14-fold increase in fluorescence intensity with a 10 nm blue shift. The fluorescence is 50% of that observed with equimolar free nucleoside, consistent with extrahelical stabilization of the target base in the enzyme-DNA complex. The shift in x further implies the base is placed into a low dielectric environment. For adenine-specific DNA methyltransferases, a hydrophobic pocket composed of highly conserved amino acids lies proximal to the cofactor binding site. Substitution of 2-aminopurine adjacent to the target base also results in detectable changes in fluorescence emission following complex formation with the methyltransferase. Thus, other classes of enzymes hypothesized to utilize base flipping can be investigated by this method.
[pdf] - Murine DNA Cytosine-C5 Methyltransferase: Pre-Steady- and Steady-State Kinetic Analysis with Regulatory DNA Sequences
James Flynn, J. Fraser Glickman, and Norbert O. Reich. Biochemistry 1996, 35, 7308-7315.We present the first description of Km DNA, Kd DNA, kcat, and kmethylation for a mammalian DNA methyltransferase. Homogeneous, 190 000 Mr DNA (cytosine-5-)-methyltransferase isolated from mouse erythroleukemia cells has turnover constants of 0.15-0.59 h-1 with single-stranded and unmethylated double-stranded oligonucleotides containing a single CpG dinucleotide. These substrates were designed to mimic DNA transcriptional cis elements previously reported to have cytosine C-5-methylated regulation. The rate-limiting step for these substrates is the methylation step itself. In contrast, hemimethylated doublestranded substrates show burst kinetics, consistent with a rapid methylation event (3 h-1) followed by a slower step which determines steady-state kcat. Hemimethylated and unmethylated double-stranded DNA shows similar binding affinities; these results reveal the molecular basis for the enzyme's preference for hemimethylated DNA to be the methyl transfer step. Substrates with multiple recognition sites do not show burst kinetics and have turnover rate constants of 6 h-1. Catalytic turnover for the mammalian enzyme is thus approximately 10-fold slower than that for the related bacterial enzymes. Our combined results show quantitatively that one enzyme is certainly capable of both maintenance and de noVo methylation and that maintenance of the genomic methylation pattern is preferred over the de noVo establishment of new patterns. Direct comparison of the mammalian enzyme with the bacterial DNA cytosine-C5 methyltransferase, M.SssI, indicates dramatic differences in preferences for single-stranded, double-stranded, and hemimethylated double-stranded substrates. Moreover, the specificity hierarchy shown for the M.SssI is derived from very different changes in Km and catalysis than those observed for the mammalian DCMTase. These results demonstrate that the M.SssI, and perhaps other DNA cytosine methyltransferases from bacteria, is functionally dissimilar to the mammalian enzyme.
[pdf] - Cofactor and DNA Interactions in EcoRI DNA Methyltransferase: Fluoresence Spectroscopy and Phenylalanine Replacement for Tryptophan 183
Karen A. Maegley, Lino Gonzalez, Jr.$, Dean W. Smith, and Norbert O. Reich. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol . 267, No. 26, Issue of September 15, PP. 18527-18532,1992.EcoRI DNA methyltransferase contains tryptophans at positions 183 and 226. Tryptophan 225 is adjacent to residues previously implicated in S-adenosylmethionine (AdoMet)b inding and tocy steine 223, previously shown to be the site of N-ethyl maleimide-mediated inactivation of the enzyme (Reich, N. O., and Everett, E. (1990) J. Biol. Chern. 265,89294934; Everett, E. A., Falick, A. M., and Reich, N. 0. (1990) J. Biol. Chern. 265, 17713-17719). The fluorescence spectra of the wild-typee nzyme is centered at 338 nm indicating partial tryptophan solventa ccessibility. Substitution of tryptophan 183 with phenylalanine results in a 46% drop inf luorescence intensity, butn o shift in X.,,, DNA binding to the wild-type methyltransferase caused an increase in thefl uorescence intensity, while binding to the tryptoph1a8n3 mutant hada quenching effect, suggesting that DNA binding induces a conformational change near both tryptophans. Binding of AdoMet and various AdoMet analogs to the wild-type methyltransferase results in no change in the fluorescence spectrum when excitation occurs at 295 nm, suggesting that no conformational change occurs, and AdoMet does not interact with either tryptophan. In contrast, quenching was observed when excitatioonc - curred at 280 nm, suggesting that AdoMet and its analogs may be quenching tyrosine to tryptophan energy transfer. Protein-ligandc omplexes were titrated with acrylamide, and the data also implicate conformational changes upon DNA binding but not upon AdoMet binding, consistent with previous limited proteolysis results (Reich, N. O., Maegley, K. A., Shoemaker, D. D., and Everett, E. (1991) Biochemistry 30, 2940-2946).
[pdf] - In Vitro Specificity of EcoRI DNA Methyltransferase
Norbert 0. Reich, Charlotte Olsen, Fabrizio Osti, and John Murphy. THE JOURNOAL OF BIOLOGICAL CHEMISTRY Vol. 267, No. 22, Issue of August 5, pp. 15802-15607,1992.The sequence selectivity of enzyme-DNA interactions was analyzed by comparing discrimination between synthetic oligonucleotides containing the canonical site GAATTC and altered DNA sequences with the EcoRI DNA methyltransferase. The specificities (kcad KENAa)r e decreased from 5- to 23,000-fold relative to the unmodified site. For several substrates the decrease in kcat makes a disproportionate contribution to the specificity difference, suggesting that discrimination is mediated by the placement of critical catalytic residues rather than binding interactions. This is supported by our observation that specificity changes are generally not followed by changes in the stability of the methyltransferase-DNA complexes. Also, base pair substitutions near the site of methylation result in greater decreases in complex stability, suggesting that recognition and catalytic mechanisms overlap.
[pdf] - Kinetic Mechanism of the EcoRI DNA Methyltransferase
Norbert 0. Reich and Neda Mashhoon. Biochemistry 1991, 30, 2933-2939.We present a kinetic analysis of the EcoRI DNA N6-adenosine methyltransferase (Mtase). The enzyme catalyzes the S-adenosylmethionine (AdoMet)-dependent methylation of a short, synthetic 14 base pair DNA substrate and plasmid pBR322 DNA substrate with k,,,/K, values of 0.51 X lo8 and 4.1 X lo8 s-l M-I, respectively. The Mtase is thus one of the most efficient biocatalysts known. Our data are consistent with an ordered bi-bi steady-state mechanism in which AdoMet binds first, followed by DNA addition. One of the reaction products, S-adenosylhomocysteine (AdoHcy), is an uncompetitive inhibitor with respect to DNA and a competitive inhibitor with respect to AdoMet. Thus, initial DNA binding followed by AdoHcy binding leads to formation of a ternary dead-end complex (Mtase-DNA-AdoHcy). We suggest that the product inhibition patterns and apparent order of substrate binding can be reconciled by a mechanism in which the Mtase binds AdoMet and noncanonical DNA randomly but that recognition of the canonical site requires AdoMet to be bound. Pre-steady-state and isotope partition analyses starting with the binary Mtase-AdoMet complex confirm its catalytic competence. Moreover, the methyl transfer step is at least 10 times faster than catalytic turnover.
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Other Files
- 1.96 A resolution WT M.HhaI ternary structure
ABSTRACT
[pdb]