Norbert Reich Biochemistry Lab

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Ben Youngblood

Research Abstract

In order to gain new insights into enzyme fidelity one aspect of my research focuses on the delicate balance between sequence specificity and efficient catalysis performed by DNA modifying enzymes. Utilization of indirect readout of a DNA sequence, such as DNA bending by an enzyme, provides further discrimination beyond the initial binding event between the enzyme and DNA (1). DNA methyltransferases incorporate the conformational transitions of DNA bending, base-flipping, and DNA intercalation into their mechanism (2), and can use such steps as checkpoints in discrimination (3). Through the use of fluorescence resonance energy transfer (FRET) and 2-AminoPurine (2AP) fluorescence studies with cognate and noncognate substrates I am examining the relationship between these transitions and how they facilitate the discrimination of the enzyme.

Another aspect of my research is examining the target selection of the mammalian methyltransferase Dnmt1. Our lab has had a long history of exploring the kinetic mechanisms of Dnmt1 (4-10). Previously other labs have shown that viral infections can redirect the mammalian methyltransferases (11;12). We are currently pursuing experiments to understand Dnmt1 target selection by introducing viral components to human cells.

References (click to expand)
  1. Johnson, K. A. (1993) Conformational coupling in DNA polymerase fidelity Annu. Rev. Biochem. 62, 685-713.
  2. Hopkins, B. B. and Reich, N. O. (2004) Simultaneous DNA binding, bending, and base flipping: Evidence for a novel M.EcoRI methyltransferase: DNA complex J. Biol. Chem.
  3. Huang, N. and MacKerell, A. D., Jr. (2005) Specificity in protein-DNA interactions: energetic recognition by the (cytosine-C5)-methyltransferase from HhaI J. Mol. Biol. 345, 265-274.
  4. Aubol, B. E. and Reich, N. O. (2003) Murine DNA cytosine C(5)-methyltransferase: in vitro studies of de novo methylation spreading Biochem. Biophys. Res. Commun. 310, 209-214.
  5. Flynn, J., Fang, J. Y., Mikovits, J. A., and Reich, N. O. (2003) A potent cell-active allosteric inhibitor of murine DNA cytosine C5 methyltransferase J. Biol. Chem. 278, 8238-8243.
  6. Flynn, J. and Reich, N. (1998) Murine DNA (cytosine-5-)-methyltransferase: steady-state and substrate trapping analyses of the kinetic mechanism Biochemistry 37, 15162-15169.
  7. Flynn, J., Glickman, J. F., and Reich, N. O. (1996) Murine DNA cytosine-C5 methyltransferase: pre-steady- and steady-state kinetic analysis with regulatory DNA sequences Biochemistry 35, 7308-7315.
  8. Glickman, J. F. and Reich, N. O. (1994) Baculovirus-mediated high level expression of a mammalian DNA methyltransferase Biochem. Biophys. Res. Commun. 204, 1003-1008.
  9. Glickman, J. F., Flynn, J., and Reich, N. O. (1997) Purification and characterization of recombinant baculovirus-expressed mouse DNA methyltransferase Biochem. Biophys. Res. Commun. 230, 280-284.
  10. Svedruzic, Z. M. and Reich, N. O. (2005) DNA cytosine C5 methyltransferase Dnmt1: catalysis-dependent release of allosteric inhibition Biochemistry 44, 9472-9485.
  11. Mikovits, J. A., Young, H. A., Vertino, P., Issa, J. P., Pitha, P. M., Turcoski-Corrales, S., Taub, D. D., Petrow, C. L., Baylin, S. B., and Ruscetti, F. W. (1998) Infection with human immunodeficiency virus type 1 upregulates DNA methyltransferase, resulting in de novo methylation of the gamma interferon (IFN-gamma) promoter and subsequent downregulation of IFN-gamma production Mol. Cell Biol. 18, 5166-5177.
  12. Robertson, K. D. (2000) The role of DNA methylation in modulating Epstein-Barr virus gene expression Curr. Top. Microbiol. Immunol. 249, 21-34.

Papers

 

Research Support

Research funding is provided by the National Science Foundation.

Presentations

 

Contact

Youngblo@lifesci.ucsb.edu

Reich Lab Contact Info