Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Checkpoints Controlled by PCNA, DNA and ATP Direct the Timing and Order of Events in the Clamp Loading Mechanism
PCNA (Proliferating Cell Nuclear Antigen) clamp is an essential ring shaped protein that is loaded around the primer-template (p/t) DNA junction to serve as a mobile tether for DNA polymerase and enhance its processivity during DNA synthesis. PCNA clamp loading is a complex process catalyzed by RFC (Replication Factor C), which belongs to AAA+ ATPase family (Sakato etal., 2012). The multi-step clamp loading reaction minimally involves: PCNA opening, p/tDNA binding, PCNA closing and release on p/tDNA by RFC coupled to ATP binding and hydrolysis (Thompson et al., 2012). Several assays have been developed to measure the transient formation/dissociation of intermediate species in the reaction, such as RFC.PCNA.ATP, RFC.PCNA.ATP.ptDNA, and their conformational transitions, in order to solve the reaction mechanism. These include p/tDNA binding and release assays, ATP hydrolysis and phosphate release assays, as well as PCNA opening/closing and release assays. Moreover, the various experiments have been performed in the presence of unlabeled or unreactive chase (e.g., unlabeled p/tDNA, non-hydrolyzable ATPγS and unlabeled PCNA), which effectively enables measurement of a single catalytic turnover and minimizes the confounding effect of steady state turnover on data interpretation. The ability to measure each step from the perspective of each substrate has allowed us to define the clamp loading mechanism in unprecedented detail. Most significantly, it has revealed rate-limiting steps that control the timing and directionality of this critically important reaction in DNA replication, repair and recombination.
This research has been supported by NSF award MCB-1022203.
Department of Molecular Biology