REPLICATION FACTOR-C INTERACTS WITH THE C-TERMINAL SIDE OF PROLIFERATING CELL NUCLEAR ANTIGEN

Replication factor C (RF-C) is a heteropentameric protein essential fo r DNA replication and repair. It is a molecular matchmaker required fo r loading of proliferating cell nuclear antigen (PCNA) onto double-str anded DNA and, thus, for PCNA-dependent DNA elongation by DNA polymera ses delta and epsilon. To elucidate the mode of RF-C binding to the PC NA clamp, modified forms of human PCNA were used that could be P-32-la beled in vitro either at the C or the N terminus. Using a kinase prote ction assay, we show that the heteropentameric calf thymus RF-C was ab le to protect the C-terminal region but not the N-terminal region of h uman PCNA from phosphorylation, suggesting that RF C interacts with th e PCNA face at which the C termini are located (C-side). A similar pro tection profile was obtained with the recently identified PCNA binding region (residues 478-712), but not with the DNA binding region (resid ues 366-477), of the human RF-C large subunit (Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messner, H., K hastilba, S., Hubscher, 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, interact ed similarly with the modified human PCNA, indicating that the interac tion between RF C and PCNA is conserved through eukaryotic evolution.