Ronald K. Gary

Professor of Biochemistry
Department of Chemistry & Biochemistry
University of Nevada, Las Vegas
4505 Maryland Parkway
Las Vegas, NV 89154-4003

Phone: (702) 895-1687


Ph.D., Biochemistry, Molecular and Cell Biology, Cornell University, 1995.
B.S., Biological Sciences, University of California, Irvine, 1983.

Major Teaching Responsibilities:

CHEM 474: Biochemistry I
CHEM 772: Nucleic Acid Chemistry
CHEM 773: Physical Biochemistry
CHEM 793: Special Topics / DNA Repair & Mutagenesis

Teaching-related publications

Gary, RK. (2004). The concentration-dependence of the ΔS term in the Gibbs free energy function:
Application to reversible reactions in biochemistry. J. Chem. Educ. 81: 1599-1604.

Research Interests:

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.

Alignment of PCNA-binding regions of FEN1, XPG, and p21 reveals key hydrophobic residues in common (bold), and suggests a similar mode of interaction with PCNA

from Gary et al., J. Biol. Chem., 1997

Discovery of the PCNA-binding motif

Common amino acid sequence features were found in 3 PCNA-binding proteins:
the DNA replication and repair endonuclease FEN1, the excision repair endonuclease XPG, and the cell cycle regulator p21 (Gary et al., J. Biol. Chem., 1997). The elucidation of these consensus elements facilitated bioinformatics-based strategies to discover sequences in additional PCNA-binding partners, such as DNA Ligase I (Montecucco et al., EMBO J., 1998), MSH3 and MSH6 (Clark et al., J. Biol. Chem., 2000). PCNA-binding activity can be selectively abolished via FF to AA site-directed mutagenesis (Gary et al., J. Biol. Chem., 1999), providing a general approach for functional studies of the interaction in various systems.

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.

mRNA Quantification
ChIP (chromatin immunoprecipitation)
using p21-specific PCR primers for analysis

In HFL-1 cells, 24 hr treatment with 10 uM Be2+ causes increased expression of one p53-regulated gene (p21) but not another (bax). p53 does not self-regulate and does not affect the expression of housekeeping genes (e.g. actin), so these remain unchanged as well.

In HFL-1 cells, p53 associates with p21 promoter DNA after 24 hr treatment with 10 uM Be2+

from Coates et al., J. Pharmacol. Exp. Ther., 2007

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).