ATP HYDROLYSIS CATALYZED BY HUMAN REPLICATION FACTOR-C REQUIRES PARTICIPATION OF MULTIPLE SUBUNITS

Human replication factor C (hRFC) is a five-subunit protein complex (p 140, p40, p38, p37, and p36) that acts to catalytically load prolifera ting cell nuclear antigen onto DNA, where it recruits DNA polymerase d elta or epsilon to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomple x of hRFC consisting of three subunits (p40, p37, and p36) contained D NA-dependent ATPase activity. However, it is not clear which subunit(s ) hydrolyzes ATP, as all five subunits include potential ATP binding s ites. In this report,,ve introduced point mutations in the putative AT P-binding sequences of each hRFC subunit and examined the properties o f the resulting mutant hRFC complex and the ATPase activity of the hRF C or the p40 p37 p36 complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replica tion activity of the hRFC complex and the ATPase activity of the hRFC or the p40 p37 p36 complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC, These find ings indicate that the replication activity of hRFC is dependent on ef ficient ATP hydrolysis contributed to by the action of four hRFC subun its.