A COMPLEX CONSISTING OF HUMAN REPLICATION FACTOR-C P40, P37, AND P36 SUBUNITS IS A DNA-DEPENDENT ATPASE AND AN INTERMEDIATE IN THE ASSEMBLYOF THE HOLOENZYME

Human replication factor C (hRFC) is a multi-subunit protein complex c apable of supporting proliferating cell nuclear antigen (PCNA) depende nt DNA synthesis by DNA polymerases delta and epsilon. The hRFC comple x consists of five different subunits with apparent molecular masses o f 140, 40, 38, 37, and 36 kDa. We have previously reported the express ion of a three-subunit core complex, consisting of the p40, p37, and p 36 subunits following coupled in vitro transcription-translation of th e cDNAs encoding these proteins (Uhlmann, F., Cai, J., Flores-Rozas, H ., Dean, F. B., Finkelstein, J., O'Donnell, M., and Hurwitz, J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6521-6526). Here we describe the isolation of a stable complex composed of the p40, p37, and p36 subuni ts of hRFC from baculovirus-infected insect cells. The purified p40.p3 7.p36 complex, like the five-subunit RFC, contained DNA-dependent ATPa se activity that was stimulated by PCNA, preferentially bound to prime d DNA templates, interacted with PCNA, and was capable of unloading PC NA from singly nicked circular DNA. In contrast to the five subunit RF C, the three-subunit core complex did not load PCNA onto DNA. The p40. p37.p36 complex inhibited the elongation of primed DNA templates catal yzed by the DNA polymerase delta holoenzyme. Incubation of the p40.p37 .p36 complex with the hRFC p140 and p38 subunits formed the five-subun it hRFC complex that supported PCNA-dependent DNA synthesis by DNA pol ymerase delta.