Abstract
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.
Publication types
-
Research Support, Non-U.S. Gov't
-
Research Support, U.S. Gov't, P.H.S.
MeSH terms
-
Adenosine Triphosphatases / metabolism
-
Animals
-
Baculoviridae
-
Cell Line
-
DNA Primers
-
DNA Replication*
-
DNA-Binding Proteins / biosynthesis*
-
DNA-Binding Proteins / chemistry*
-
DNA-Binding Proteins / isolation & purification
-
HeLa Cells
-
Homeodomain Proteins*
-
Humans
-
Kinetics
-
Macromolecular Substances
-
Minor Histocompatibility Antigens
-
Proliferating Cell Nuclear Antigen / analysis
-
Proto-Oncogene Proteins c-bcl-2*
-
Recombinant Proteins / biosynthesis
-
Recombinant Proteins / chemistry
-
Recombinant Proteins / isolation & purification
-
Replication Protein C
-
Repressor Proteins*
-
Saccharomyces cerevisiae Proteins*
-
Spodoptera
-
Transfection
Substances
-
BCL2-related protein A1
-
DNA Primers
-
DNA-Binding Proteins
-
Homeodomain Proteins
-
MATA1 protein, S cerevisiae
-
Macromolecular Substances
-
Minor Histocompatibility Antigens
-
Proliferating Cell Nuclear Antigen
-
Proto-Oncogene Proteins c-bcl-2
-
RFC2 protein, human
-
RFC3 protein, human
-
RFC4 protein, human
-
Recombinant Proteins
-
Repressor Proteins
-
Saccharomyces cerevisiae Proteins
-
Adenosine Triphosphatases
-
Replication Protein C