Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

Kazunari Hashiguchi, Yoshihiro Matsumoto, Akira Yasui

    Research output: Contribution to journalArticlepeer-review

    33 Citations (Scopus)

    Abstract

    The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.

    Original languageEnglish
    Pages (from-to)2913-2923
    Number of pages11
    JournalNucleic acids research
    Volume35
    Issue number9
    DOIs
    Publication statusPublished - 2007 May

    ASJC Scopus subject areas

    • Genetics

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