Activities and Awards
Beckman Young Investigator Award, 1993-1996
A.P. Sloan Fellow 1998-2000
Member, NIH Bioorganic and Natural Products Study Section 2002-2007
The research in my laboratory focuses on the fascinating area of DNA repair. Damage to DNA can result in deleterious outcomes, such as cancer and aging; however, fortunately, most DNA damage is repaired by DNA repair enzymes. Oxidative damage to guanine leads to the formation of significant amounts of OG (where OG = 7,8-dihydro-8-oxoguanine) which promotes misincorporation of A during DNA replication to form OG:A base-pairs. In E. coli , two enzymes prevent mutations caused by OG; the Fpg protein removes OG from OG:C base pairs, while the MutY enzyme removes adenine from OG:A base pairs. Once the damaged or inappropriate bases are removed, they are replaced with the normal undamaged bases. The goals of our research are to understand the molecular details associated with the recognition and repair of oxidative DNA damage by MutY and Fpg, as well as related human and yeast homologues. In the process of reaching these goals, we are developing novel methods for investigating DNA-protein interactions.
Figure 1: DNA damage can lead to cancer and
aging; fortunately, in most cases, DNA damaged is repaired!
Our approach to elucidating the properties of DNA damage recognition and repair by DNA repair enzymes is multifaceted, involving many different techniques. The tools of enzymology (protein purification, kinetics, etc.) are used to study the enzyme and its interactions with DNA substrates. Synthetic techniques are used to synthesize modified DNA substrates (Fig. 2) to test hypotheses about mechanism. Biophysical techniques are used to characterize the protein-DNA complex. We also use the tools of molecular biology in the overproduction and site-directed mutagenesis.
The multidisciplinary nature of this type of research on the interface between chemistry and biology is demanding yet exciting and rewarding. I believe that the forte of this research program is the use of a diverse group of approaches involving oligonucleotide chemistry, enzymology and physical methods to
address important molecular questions in the field of DNA repair. In the process, understanding of a complex and interesting biological process will unfold, and new and interesting chemistry will be discovered.
Figure 2: Substitutions of "A" in an OG:A mismatch substrates and the relative decrease in activity with MutY. This study provided insight into features important for recognition and removal of adenine opposite OG.
- Chepanoske, C.L., et al ."A Residue in MutY Important for Catalysis Identified by Photocross-Linking and Mass Spectrometry,"Biochemistry , 43 , 651-662 ( 2004 ).
- Chmiel, N.H., Livingston, A.L., David, S.S. "Insight into the Functional Consequences of Inherited Variants of MYH associated with Colorectal Cancer,"J. Mol. Biol . 327 , 431-443 ( 2003 ).
- Leipold, M.D., et al., "Recognition and Removal of Oxidized Guainines in Duplex DNA by Base Excision Repair Enzymes hOGG1, yOGG1 and yOGG2," Biochemistry , 42 , 11373-11381 ( 2003 ).
- Francis, A. W., et al., "Probing the Requirements for Recognition and Catalysis in Fpg and MutY with Nonpolar Adenine Isosteres,"J. Am. Chem. Soc. , 125 , 16235-16242 ( 2003 ).
- Francis, A.W., David, S.S. " Escherichia coli MutY and Fpg Utilize a Processive Mechanism for Target Location," Biochemistry , 42 , 801-810 ( 2003 ).