TY - JOUR
T1 - Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells
AU - Hashiguchi, Kazunari
AU - Matsumoto, Yoshihiro
AU - Yasui, Akira
N1 - Funding Information:
We are grateful to Samuel Wilson (NIEHS/NIH) for providing the wild-type and xrcc1-deficient cell lines. This work was supported in part by a grant of the Genome Network Project and by Grant-in-Aid for Scientific Research 18012003 from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to A.Y.). Funding to pay the Open Access publication charge was provided by the Genome Network Project.
PY - 2007/5
Y1 - 2007/5
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/gkm115
DO - 10.1093/nar/gkm115
M3 - Article
C2 - 17439963
AN - SCOPUS:34250634072
SN - 0305-1048
VL - 35
SP - 2913
EP - 2923
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 9
ER -