Replication factor C disengages from proliferating cell nuclear antigen (PCNA) upon sliding clamp formation, and PCNA itself tethers DNA polymerase delta to DNA.
Replication factor C (RF-C) and proliferating cell nuclear antigen (PCNA) assemble a complex, called sliding clamp, onto DNA. The clamp in turn loads DNA polymerases (pol) delta and epsilon to form the corresponding holoenzymes, which play an essential role in replication of eukaryotic chromosomal DNA and in several DNA repair pathways. To determine the fate of RF-C after loading of PCNA onto DNA, we tagged the RF-C subunit p37 with a protein kinase A recognition motif, so that the recombinant five-subunit RF-C complex could be 32P-labeled and quantitatively detected in femtomolar amounts. Nonspecific binding of RF-C to DNA was minimized by replacing the p140 subunit with an N-terminally truncated p140 subunit lacking the previously identified nonspecific DNA binding domain. Neither of these modifications impaired the clamp loading activity of the recombinant RF-C. Using gel filtration techniques, we demonstrated that RF-C dissociated from the DNA after clamp loading or pol delta holoenzyme assembly, while PCNA or PCNA.pol delta complex remained bound to DNA. PCNA catalytically loaded onto the template-primer was sufficient by itself to tether pol delta and stimulate DNA replication. The readdition of RF-C to the isolated PCNA.DNA complex did not further stimulate pol delta DNA synthesis. We conclude that pol delta holoenzyme consists of PCNA and pol delta core and that RF-C serves only to load PCNA clamp.
Podust
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VN
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235 and Vanderbilt Cancer Center, Nashville, Tennessee 37232-6838, USA.
Tiwari
N
N
Stephan
S
S
Fanning
E
E
D000818:Animals; D002417:Cattle; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004258:DNA Polymerase III / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018398:Homeodomain Proteins; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D016133:Polymerase Chain Reaction; D018809:Proliferating Cell Nuclear Antigen / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology; 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; C497563:RFC4 protein, human; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004258:DNA Polymerase III; D051818:Replication Protein C