Replication factor C interacts with the C-terminal side of proliferating cell nuclear antigen

J Biol Chem. 1997 Jan 17;272(3):1769-76. doi: 10.1074/jbc.272.3.1769.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cattle
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • DNA Replication
  • DNA-Binding Proteins / metabolism*
  • Drosophila melanogaster
  • Homeodomain Proteins*
  • Humans
  • Minor Histocompatibility Antigens
  • Phosphorylation
  • Proliferating Cell Nuclear Antigen / metabolism*
  • Protein Binding
  • Proto-Oncogene Proteins c-bcl-2*
  • Replication Protein C
  • Repressor Proteins*
  • Saccharomyces cerevisiae Proteins*
  • Thymus Gland / metabolism

Substances

  • BCL2-related protein A1
  • DNA-Binding Proteins
  • Homeodomain Proteins
  • MATA1 protein, S cerevisiae
  • Minor Histocompatibility Antigens
  • Proliferating Cell Nuclear Antigen
  • Proto-Oncogene Proteins c-bcl-2
  • RFC5 protein, human
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • Cyclic AMP-Dependent Protein Kinases
  • Replication Protein C

Associated data

  • GENBANK/U97685