 | Alex Drohat,
Ph.D.
Assistant Professor
Biochemistry and Molecular Biology
School of Medicine
410-706-8118
adroh001@umaryland.edu
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ResearchWe use NMR spectroscopy and a variety of other biophysical, biochemical, and molecular biological approaches to determine the structure and elucidate mechanism of DNA repair enzymes. The reactive nucleobases of DNA are continuously damaged (chemically modified) by cellular metabolites and exogenous agents, producing cytotoxic and/or mutagenic lesions that play a role in the development of disease and in ageing. Counteracting this inevitable threat is the base excision repair (BER) pathway, initiated by a damage-specific DNA glycosylase. Using a base-flipping mechanism, these enzymes find and remove damaged and mismatched bases in the vast expanse of normal DNA. While some DNA glycosylases exhibit significant catalytic power, they are perhaps more impressive for their extraordinary specificity for certain lesions and against normal bases. Some DNA glycosylases recognize a single lesion, whereas others are more permissive and can remove multiple forms of damage. We are studying two human DNA glycosylases that are specific for G/T and G/U mispairs in addition to numerous other lesions. A central question we are addressing is how these enzymes achieve specificity for a broad range of lesions while avoiding normal bases. We are also investigating the general question of how the activity of DNA glycosylases is stimulated by AP endonuclease, the follow-on enzyme in BER, i.e. how are the first two steps of BER coupled? Finally, we are interested in characterizing and understanding the biological role of protein-protein interactions among BER enzymes, and involving BER enzymes and proteins from other pathways, many of which are involved in regulating gene expression.
Lab TechniquesEnzyme kinetics (steady-state and pre-steady-state), NMR spectroscopy, X-ray crystallography, fluorescence spectroscopy, isothermal titration calorimetry, structure activity relationships, site-directed mutagenesis, protein and nucleic acid purification, electrophoretic mobility shift assay (EMSA), analytical HPLC
PublicationsSELECTED PUBLICATIONS (all references in PubMed)
Fitzgerald ME and Drohat AC (2008) Coordinating the Initial Steps of Base Excision Repair: AP Endonuclease 1 Actively Stimulates Thymine DNA Glycosylase by Disrupting the Product Complex, J. Biol. Chem., published online 9-19-2008
Maiti A, Morgan MT, Pozharski E, and Drohat AC (2008) Crystal Structure of Human Thymine DNA Glycosylase Bound to DNA Elucidates Sequence-Specific Mismatch Recognition, Proc. Natl. Acad. Sci. U.S.A. 105, 8890-8895
Fitzgerald ME, and Drohat AC (2008) Structural Studies of RNA/DNA Polypurine Tracts. Chem. Biol. 15, 203-204. (invited commentary)
Morgan MT, Bennett MT, and Drohat AC (2007) Excision of 5-Halogenated Uracils by Human Thymine DNA Glycosylase: Robust Activity for DNA Contexts Other than CpG. J. Biol. Chem. 282, 27578-27586
Guan X, Madabushi A, Chang DY, Fitzgerald ME, Shi G, Drohat, AC, Lu, AL (2007) The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates DNA repair enzyme TDG glycosylase, Nucleic Acids Res. 35, 6207-6218
Bennett MT, Rodgers MT, Hebert AS, Ruslander LE, Eisele L, and Drohat AC (2006) Specificity of Human Thymine DNA Glycosylase Depends on N-Glycosidic Bond Stability, J. Am. Chem. Soc. 128, 12510-12519
Cao C, Kwon K, Jiang YL, Drohat AC, and Stivers JT (2003) Solution structure and base perturbation studies reveal a novel mode of alkylated base recognition by 3-methyladenine DNA glycosylase I, J. Biol. Chem. 278, 48012-48020
Jiang YL, Drohat AC, Ichikawa Y, and Stivers JT (2002) Probing the Limits of Electrostatic Catalysis by Uracil DNA Glycosylase Using Transition-State Mimicry and Mutagenesis, J. Biol. Chem. 277, 15385-15392
Drohat AC, Kwon K, Krosky DL, and Stivers JT (2002) 3‑methyladenine DNA Glycosylase I is an Unexpected Helix-Hairpin-Helix Superfamily Member, Nature Struct. Biol. 9, 659-664. This paper featured in News and Views.
Drohat AC, and Stivers JT (2000) NMR Evidence for an Unusually Low N1 pKa for Uracil Bound to Uracil DNA Glycosylase: Implications for Catalysis, J. Am. Chem. Soc. 122, 1840-1841
Drohat AC, and Stivers JT (2000) Escherichia coli Uracil DNAGlycosylase: NMR Characterization of the Short Hydrogen Bond from His187 to Uracil O2, Biochemistry 39, 11865-11875 Personal HistoryEDUCATION
1988 - B.S., Aerospace Engineering, University of Maryland College Park
1997 - Ph.D., Biochemistry and Molecular Biology, University of Maryland Baltimore
POST GRADUATE EXPERIENCE
1998-2000 National Research Council Postdoctoral Fellow, Center for Advanced Research in Biotechnology, Rockville MD
2001-2002 Postdoctoral Fellow, Johns Hopkins University School of Medicine Laboratory PersonnelBrittney Manvilla (graduate student)
Megan Fitzgerald (graduate student)
Atanu Maiti, PhD (postdoctoral fellow)
Michael Morgan (graduate student) Other Resources Department of Biochemistry and Molecular Biology
I am a member of the Molecular and Structural Biology Program, Greenebaum Cancer Center
NMR Facility at UMB
X-ray Crystallography at UMB
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