Replication factor C interacts with the C-terminal side of proliferating cell nuclear antigen.
Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and repair. It is a molecular matchmaker required for loading of proliferating cell nuclear antigen (PCNA) onto double-stranded DNA and, thus, for PCNA-dependent DNA elongation by DNA polymerases delta and epsilon. To elucidate the mode of RF-C binding to the PCNA clamp, modified forms of human PCNA were used that could be 32P-labeled in vitro either at the C or the N terminus. Using a kinase protection assay, we show that the heteropentameric calf thymus RF-C was able to protect the C-terminal region but not the N-terminal region of human PCNA from phosphorylation, suggesting that RF-C interacts with the PCNA face at which the C termini are located (C-side). A similar protection profile was obtained with the recently identified PCNA binding region (residues 478-712), but not with the DNA binding region (residues 366-477), of the human RF-C large subunit (Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messner, H., Khastilba, S., Hübscher, U., and Fotedar, A., (1996) EMBO J., 15, 4423-4433). Furthermore, we show that the RF-C 36 kDa subunit of human RF-C could interact independently with the C-side of PCNA. The RF-C large subunit from a third species, namely Drosophila melanogaster, interacted similarly with the modified human PCNA, indicating that the interaction between RF-C and PCNA is conserved through eukaryotic evolution.
Mossi
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R
Institute of Veterinary Biochemistry, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Jónsson
Z O
ZO
Allen
B L
BL
Hardin
S H
SH
Hübscher
U
U
D000818:Animals; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster; D018398:Homeodomain Proteins; D006801:Humans; D015778:Minor Histocompatibility Antigens; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013950:Thymus Gland / Q000378:metabolism
C096816:BCL2-related protein A1; 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; C497564:RFC5 protein, human; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D051818:Replication Protein C