Nucleic Acids Research Advance Access originally published online on April 16, 2007
Nucleic Acids Research 2007 35(9):2913-2923; doi:10.1093/nar/gkm115
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Nucleic Acids Research, 2007, Vol. 35, No. 9 2913-2923
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells
1Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan and 2Division of Medical Science, Fox Chase Cancer Center, PA 19111, USA
*To whom correspondence should be addressed. Tel: +81-22-717-8465; Fax: +81-22-717-8470; Email: ayasui{at}idac.tohoku.ac.jp Correspondence may also be addressed to Kazunari Hashiguchi. Tel: +81-22-717-8469; Fax: +81-22-717-8470; Email: hashiguchika{at}idac.tohoku.ac.jp
Received December 6, 2006. Revised February 3, 2007. Accepted February 8, 2007.
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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