Journal
NUCLEIC ACIDS RESEARCH
Volume 45, Issue 8, Pages 4550-4563Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkx096
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Funding
- KAKENHI [25131714, 25440011, 26114714, 16H04743]
- JSPS (KAKENHI) [16H04743]
- Grants-in-Aid for Scientific Research [16H06306, 25440011, 26114714, 16H04743] Funding Source: KAKEN
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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.
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