Regulation of WRN Deacetylation by SIRT1 after DNA Damage

Dr. Orren's lab has been involved in the biochemical characterization of WRN and other RecQ helicases for more than 10 years and published extensively in this area. For this project, the Orren laboratory will be determining the effects of WRN acetylation on its enzymatic properties in order to understand how this modification and its reversal impacts the DNA metabolic function of WRN. These experiments involve characterization and comparison the enzymatic activities of unmodified, acetylated, and deacetylated WRN preparations on a variety of DNA substrates. Specifically, unmodified WRN possesses ATPase, DNA unwinding (helicase), exonuclease, annealing, and branch migration activities, and its physiological DNA substrates are likely to be replication or recombination intermediates. During the current period, the DNA binding, helicase, exonuclease, and annealing activities of unmodified and acetylated WRN on DNA structures reflecting replication fork, Holliday junction, and strand invasion intermediates will be directly compared. The lab will also examine mutant WRN proteins that cannot be acetylated or can serve as acetylation mimics to confirm correct folding and normal helicase and exonuclease activities. Furthermore, the lab will assist Dr. Luo in investigating the dynamics of WRN acetylation and deacetylation in cells, the relationship of these events to the induction and persistence of DNA damage and to the cell cycle. Graduate student Enerlyn Lozada will be responsible for performing these experiments under Dr. Orren's supervision at the University of Kentucky College of Medicine.