A complex consisting of human replication factor C p40, p37, and p36 subunits is a DNA-dependent ATPase and an intermediate in the assembly of the holoenzyme.
Human replication factor C (hRFC) is a multi-subunit protein complex capable of supporting proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis by DNA polymerases delta and epsilon. The hRFC complex consists of five different subunits with apparent molecular masses of 140, 40, 38, 37, and 36 kDa. We have previously reported the expression of a three-subunit core complex, consisting of the p40, p37, and p36 subunits following coupled in vitro transcription-translation of the 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 subunits of hRFC from baculovirus-infected insect cells. The purified p40.p37.p36 complex, like the five-subunit RFC, contained DNA-dependent ATPase activity that was stimulated by PCNA, preferentially bound to primed DNA templates, interacted with PCNA, and was capable of unloading PCNA from singly-nicked circular DNA. In contrast to the five-subunit RFC, the three-subunit core complex did not load PCNA onto DNA. The p40. p37.p36 complex inhibited the elongation of primed DNA templates catalyzed by the DNA polymerase delta holoenzyme. Incubation of the p40.p37.p36 complex with the hRFC p140 and p38 subunits formed the five-subunit hRFC complex that supported PCNA-dependent DNA synthesis by DNA polymerase delta.
Cai
J
J
Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Gibbs
E
E
Uhlmann
F
F
Phillips
B
B
Yao
N
N
O'Donnell
M
M
Hurwitz
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002455:Cell Division; D004265:DNA Helicases; D004258:DNA Polymerase III; D004261:DNA Replication; D004270:DNA, Circular / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D015778:Minor Histocompatibility Antigens; D008970:Molecular Weight; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D018411:Spodoptera
C096816:BCL2-related protein A1; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; C497561:RFC2 protein, human; C497562:RFC3 protein, human; C497563:RFC4 protein, human; C497564:RFC5 protein, human; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D051818:Replication Protein C