||Ryo Fujisawa, Eiji Ohashi, Kouji Hirota, Toshiki Tsurimoto, Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ϵ efficiently loads the PCNA sliding clamp, Nucleic acids research, 10.1093/nar/gkx096, 45, 8, 4550-4563, 2017.01, The alternative proliferating-cell nuclear antigen (PCNA)-loader CTF18-RFC forms a stable complex with DNA polymerase ϵ (Polϵ). We observed that, under near-physiological conditions, CTF18- RFC alone loaded PCNA inefficiently, but loaded it efficiently when complexed with Polϵ. During efficient PCNA loading, CTF18-RFC and Polϵ assembled at a 3ϵ primer-template junction cooperatively, and directed PCNA to the loading site. Site-specific photo-crosslinking of directly interacting proteins at the primer-template junction showed similar cooperative binding, in which the catalytic N-terminal portion of Polϵ acted as the major docking protein. In the PCNA-loading intermediate with ATPαS, binding of CTF18 to the DNA structures increased, suggesting transient access of CTF18-RFC to the primer terminus. Polϵ placed in DNA synthesis mode using a substrate DNA with a deoxidised 3ϵ primer end did not stimulate PCNA loading, suggesting that DNA synthesis and PCNA loading are mutually exclusive at the 3ϵ primer-template junction. Furthermore, PCNA and CTF18-RFC-Polϵ complex engaged in stable trimeric assembly on the template DNA and synthesised DNA efficiently. Thus, CTF18-RFC appears to be involved in leading-strand DNA synthesis through its interaction with Polϵ, and can load PCNA onto DNA when Polϵ is not in DNA synthesis mode to restore DNA synthesis..
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