Mechanism underlying replication protein a stimulation of DNA ligase I.
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 is a central DNA repair pathway, responsible for the removal of damaged bases. We have shown previously 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 base excision repair component human DNA ligase I by approximately 15-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 k(cat) is observed, whereas K(m) 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.
Ranalli
Tamara A
TA
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
DeMott
Michael S
MS
Bambara
Robert A
RA
D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D002384:Catalysis; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000502:physiology*; D004789:Enzyme Activation; D007700:Kinetics; D051716:Replication Protein A
D017931:DNA Primers; D004268:DNA-Binding Proteins; C000607892:LIG1 protein, human; D051716:Replication Protein A; D011088:DNA Ligases; D000072481:DNA Ligase ATP