Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells.
Human replication factor C (RFC, also called activator 1) is a five-subunit protein complex (p140, p40, p38, p37, and p36) required for proliferating cell nuclear antigen (PCNA)-dependent processive DNA synthesis catalyzed by DNA polymerase delta or epsilon. Here we report the reconstitution of the RFC complex from its five subunits simultaneously overexpressed in baculovirus-infected insect cells. The purified baculovirus-produced RFC appears to contain equimolar levels of each subunit and was shown to be functionally identical to its native counterpart in (i) supporting DNA polymerase delta-catalyzed PCNA-dependent DNA chain elongation; (ii) catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and human single-stranded DNA binding protein; (iii) binding preferentially to DNA primer ends; and (iv) catalytically loading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.
Cai
J
J
Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Uhlmann
F
F
Gibbs
E
E
Flores-Rozas
H
H
Lee
C G
CG
Phillips
B
B
Finkelstein
J
J
Yao
N
N
O'Donnell
M
M
Hurwitz
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D017931:DNA Primers; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D018411:Spodoptera; D014162:Transfection
C096816:BCL2-related protein A1; D017931:DNA Primers; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D046911:Macromolecular Substances; 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; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; D051818:Replication Protein C