Impact of Individual PCNA-DNA Contacts on Clamp Loading and Function on DNA

Abstract

Ring-shaped clamp proteins encircle DNA and affect the work of many proteins, notably processive replication by DNA polymerases. Crystal structures of clamps show several cationic residues inside the ring, and in a co-crystal of E. coli β clamp-DNA they directly contact the tilted duplex passing through. To investigate the role of these contacts in reactions involving circular clamps, we examined single arginine/lysine mutants of S. cerevisiae PCNA in RFC-catalyzed loading of the clamp onto primer-template DNA (ptDNA). Previous kinetic analysis has shown that ptDNA entry inside an ATP-activated RFC-PCNA complex accelerates clamp opening and ATP hydrolysis, which is followed by slow PCNA closure around DNA and product dissociation. Here we directly measured multiple steps in the reaction--PCNA opening, ptDNA binding, PCNA closure, phosphate release and complex dissociation to determine if mutation of PCNA residues R14, K20, R80, K146, R149 or K217 to alanine affects the reaction mechanism. Contrary to earlier steady state analysis of these mutants, pre-steady state data show that loss of single cationic residues can alter the rates of all DNA-linked steps in the reaction as well as movement of PCNA on DNA. These results explain an earlier finding that individual arginines and lysines inside human PCNA are essential for pol δ processivity. Mutations in the N-terminal domain have greater impact than in the C-terminal domain, indicating a positional bias in PCNA-DNA contacts that can influence its functions on DNA.