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Papers In Press, published online ahead of print November 6, 2001
Department of Biochemistry & Biophysics, University of Rochester School of Medicine & Dentistry, Rochester, NY 14642
Corresponding Author: robert_bambara{at}urmc.rochester.edu
Replication protein A (RPA) is a heterotrimeric single-stranded DNA binding protein that participates in multiple DNA transactions that include replication and repair. Base excision repair (BER) is a central DNA repair pathway, responsible for the removal of damaged bases. We have previously shown that RPA was able to stimulate long-patch base excision repair reconstituted in vitro. Herein we show that human RPA stimulates the activity of the BER component human DNA ligase I by approximately15-fold. Other analyzed single-stranded binding proteins would not substitute, attesting to the specificity of the stimulation. Conversely, RPA was unable to stimulate the functionally homologous ATP-dependent ligase from T4 bacteriophage. Kinetic analyses suggest that catalysis of ligation is enhanced by RPA, as a 4-fold increase in kcat is observed, while Km is not significantly changed. Substrate competition experiments further support the conclusion that RPA does not alter the specificity or rate of substrate binding by DNA ligase I. Additionally, RPA is unable to significantly enhance ligation on substrates containing an unannealed 3’-upstream primer terminus, suggesting that RPA does not stabilize the nick site to enhance ligase recognition. Furthermore when DNA ligase I is pre-bound to the substrate and limited to a single-turnover, RPA is still able to stimulate ligation. Overall, the results support a mechanism of stimulation that involves increasing the rate of catalysis of ligation.
J. Biol. Chem, 10.1074/jbc.M109053200
Submitted on September 19, 2001
Revised on October 31, 2001
Accepted on November 6, 2001
Mechanism underlying replication protein A stimulation of DNA Ligase I
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