Major Teaching Responsibilities:
CHEM 474: Biochemistry I
Gary, RK. (2004). The concentration-dependence of the ΔS term in the Gibbs free energy function:
I. DNA Damage Response Signaling & DNA Repair
We are interested in cellular responses to DNA damage and the biochemistry of DNA repair. Human cells are capable of repairing most types of DNA damage. This process provides a critical natural defense against mutation and cancer. We are investigating the interactions between repair endonucleases, which remove damaged DNA, and proliferating cell nuclear antigen (PCNA), which helps to synthesize new (undamaged) DNA at the repair site. Other kinds of proteins can also bind to PCNA, including mismatch recognition complexes (e.g. MSH2-MSH6) and cell cycle regulatory proteins (such as p21, a cyclin-dependent kinase inhibitor). A major goal of the laboratory is to learn how these regulatory and repair proteins work together as part of an integrated cellular response to DNA damage. A related project examines the function and regulation of the human tumor suppressor p53. When DNA damage occurs, p53 coordinates various protective responses, such as transient cell cycle arrest or apoptosis.
Discovery of the PCNA-binding motif
Common amino acid sequence features were found in 3 PCNA-binding proteins:
Analysis of protein-protein interactions provides insight into how enzymes work together during DNA repair. Additional interactions of interest to the lab include FEN1-WRN (Sharma et al., Nucleic Acids Res., 2005), and FEN1-NEIL1 (Hegde et al., J. Biol. Chem., 2008).
PCNA subunits (magenta, grey, brown) form a trimer that can encircle DNA (image adapted from PDB 1AXC, deposited by Gulbis & Kuriyan). PCNA-binding peptides (yellow, gold, green) make a tight complex with PCNA. An expanded view of the p21 peptide (yellow) is shown with selected H-bond constraints that define a 310 helix conformation (modeled with InsightII using Nevada INBRE Structural Bioinformatics Core workstation).
II. Cell Cycle Regulation & Inducible Senescence
Cellular senescence is a cytostatic state that is initiated by telomere shortening. During senescence, the p53 transcription factor becomes active, and causes p21 to be expressed at high levels. The transcriptional activity of p53 is modulated by post-translational modifications and interactions with binding partners. As a result, different sets of genes may be transcribed by p53 under different circumstances. For example, p53 controls the expression of bax (a pro-apoptotic protein) and p21 (a regulator of cell cycle arrest and senescence), but these products can be expressed selectively (see left panel below). We have found that Be2+ induces a senescence-like response in human fibroblasts.
A recently-developed quantitative assay (Gary and Kindell, Analytical Biochem., 2005) for measuring senescence-associated beta-galactosidase activity (SA-B-gal) shows that Be2+ treatment induces the expression of this enzymatic biomarker of cellular senescence (Coates et al., J. Pharmacol. Exp. Ther., 2007).
Cover image from The Journal of Pharmacology and Experimental Therapeutics, July 2007. Schematic of the hypothesis that Be2+ targets an upstream regulator of p53 to induce a senescence-like response. See article for details (Coates et al., J. Pharmacol. Exp. Ther., 2007).