A second proliferating cell nuclear antigen loader complex, Ctf18-replication factor C, stimulates DNA polymerase η activity

Abstract

Replication factor C (RFC) loads the clamp protein PCNA ontoDNAstructures. Ctf18-RFC, which consists of the chromosome cohesion factors Ctf18, Dcc1, and Ctf8 and four small RFC subunits, functions as a second proliferating cell nuclear antigen (PCNA) loader. To identify potential targets of Ctf18-RFC, human cell extracts were assayed for DNA polymerase activity specifically stimulated by Ctf18-RFC in conjunction with PCNA. After several chromatography steps, an activity stimulated by Ctf18-RFC but not by RFC was identified. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis revealed the presence of two DNA polymerases, η and λ, in the most purified fraction, but experiments with purified recombinant proteins demonstrated that only polymerase (pol) η was responsible for activity. Ctf18-RFC alone stimulated pol η, and the addition of PCNA cooperatively increased stimulation. Furthermore, Ctf18-RFC interacted physically with pol η, as indicated by co-precipitation in human cells. We propose that this novel loader-DNA polymerase interaction allows DNA replication forks to overcome interference by various template structures, including damaged DNA and DNA-protein complexes that maintain chromosome cohesion. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.