Cryo- EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC

• 成果类型: Article
• 署名作者: He, Qing; Wang, Feng; O'Donnell, Michael E.; Li, Huilin
• 署名单位: Van Andel Institute; Rockefeller University; Howard Hughes Medical Institute; Rockefeller University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13487
• DOI: 10.1073/pnas.2319727121
• 发表日期: 2024-04-30
• 关键词: sister-chromatid cohesion; dna-polymerase clamp; structural-analysis; replication; binding; establishment; RECRUITMENT; atpases

摘要

The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well - studied pentameric ATPase complex RFC (RFC1 - 5). The CTF18RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase epsilon and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo - EM analysis of in vitro assembled human CTF18RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3 '- ss/dsDNA junction by CTF18RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique beta- hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3 '- end of the primer; this ensures its ability to load PCNA to any 3 '- ss/dsDNA junction, facilitated by the binding energy of the E - plug to the major groove. Our study reveals unique structural features of the human CTF18RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders.