The subunits of activator 1 (replication factor C) carry out multiple functions essential for proliferating-cell nuclear antigen-dependent DNA synthesis.
p37 and p40 are two cloned gene products of the five-subunit human cellular DNA replication factor activator 1 (A1) protein complex (also called replication factor C). Here, we describe the solubilization, purification, and characterization of these two proteins that were overproduced in Escherichia coli. Using a nitrocellulose filter binding assay, we demonstrated that the purified A1 p37 protein associated with DNA preferentially at the primer terminus, a property resembling that of the A1 complex. We also show that in the presence of relatively high levels of salt, the recombinant p37 protein alone activated DNA polymerase epsilon but not polymerase delta in catalyzing the elongation of DNA chains. The p40 protein specifically associated with cellular p37 and proliferating-cell nuclear antigen (PCNA) present in HeLa cell cytosolic extract. The addition of purified p40 protein abolished the in vitro polymerase delta-catalyzed DNA elongation reaction dependent on both PCNA and A1. However, this inhibition was reversed by excess polymerase delta, suggesting a specific interaction between the polymerase and the p40 protein. Thus, while p37 binds DNA at the primer end and has a specific affinity for pol epsilon, p40, which binds ATP, interacts with PCNA and pol delta. These activities are essential for the DNA elongation reactions that lead to the synthesis of leading-strand DNA and the maturation of Okazaki fragments.
Pan
Z Q
ZQ
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Chen
M
M
Hurwitz
J
J
D001324:Autoantigens / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004258:DNA Polymerase III; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D009687:Nuclear Proteins / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins
D001324:Autoantigens; C096816:BCL2-related protein A1; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; C497561:RFC2 protein, human; C497563:RFC4 protein, human; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D051818:Replication Protein C