Replication Protein A Prohibits Diffusion of the PCNA Sliding Clamp along Single-Stranded DNA

• DOI: 10.1021/acs.biochem.6b01213
• Publication Date: 2017-02-08T18:37:52Z
• Authors: Mark Hedglin; Stephen J. Benkovic
• Keywords: DNA clamp; Replication protein A; Primer (cosmetics); DNA polymerase delta; Replication factor C

The replicative polymerases cannot accommodate distortions to the native DNA sequence such as modifications (lesions) template bases from exposure reactive metabolites and environmental mutagens. Consequently, synthesis on an afflicted abruptly stops upon encountering these lesions, but replication fork progresses onward, exposing long stretches of damaged before eventually stalling. Such arrests may be overcome by translesion (TLS) in which specialized TLS bind resident proliferating cell nuclear antigen (PCNA) replicate DNA. Hence, a critical aspect is maintaining PCNA at or near blocked primer/template (P/T) junction uncoupling progression polymerases. single-stranded (ssDNA) binding protein, protein A (RPA), coats exposed might prohibit diffusion along adjacent P/T junction. However, this idea had yet directly tested. We recently developed unique Cy3-Cy5 Forster resonance energy transfer (FRET) pair that reports occupancy PCNA. In study, we utilized FRET continuously monitor retention human Results extensive steady state pre-steady assays indicate RPA binds tightly ssDNA restricts upstream duplex region physically blocking ssDNA.