Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ε efficiently loads the PCNA sliding clamp

The alternative proliferating-cell nuclear antigen (PCNA)-loader CTF18-RFC forms a stable complex with DNA polymerase epsilon (Pol epsilon). We observed that, under near-physiological conditions, CTF18-RFC alone loaded PCNA inefficiently, but loaded it efficiently when complexed with Pol epsilon. During efficient PCNA loading, CTF18-RFC and Pol epsilon assembled at a 3' 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 Pol epsilon acted as the major docking protein. In the PCNA-loading intermediate with ATP gamma S, binding of CTF18 to the DNA structures increased, suggesting transient access of CTF18-RFC to the primer terminus. Pol epsilon placed in DNA synthesis mode using a substrate DNA with a deoxidised 3' primer end did not stimulate PCNA loading, suggesting that DNA synthesis and PCNA loading are mutually exclusive at the 3' primer-template junction. Furthermore, PCNA and CTF18-RFC-Pol epsilon 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 Pol epsilon, and can load PCNA onto DNA when Pol epsilon is not in DNA synthesis mode to restore DNA synthesis.