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J. Biol. Chem., Vol. 283, Issue 14, 9071-9079, April 4, 2008

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DNA Damage-induced Ubiquitylation of RFC2 Subunit of Replication Factor C Complex^*

Junya Tomida, Yuji Masuda^¶, Hidekazu Hiroaki^||, Tomoko Ishikawa^**, Ihnyoung Song, Toshiki Tsurimoto, Satoshi Tateishi^¶¶, Tadahiro Shiomi^||||, Yasuhiro Kamei^**, Jinhyeong Kim^**, Kenji Kamiya^¶, Cyrus Vaziri, Haruo Ohmori, and Takeshi Todo^**1

From the Radiation Biology Center, Kyoto University, Kyoto 606-8501, Japan, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan, ^¶Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan, ^||Division of Structural Biology, Department of Biotechnology and Molecular Biology, Graduate School of Medicine, Kobe University, Kobe 650-0017, Japan, ^**Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan, Department of Genetics and Genomics, Boston University School of Medicine, Boston, Massachusetts 02118, Department of Biology, School of Science, Kyushu University, Fukuoka 812-8581, Japan, ^¶¶Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan, and ^||||National Institute of Radiological Science, Chiba 263-8555, Japan

Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono- and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H₂O₂. A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.

Received for publication, December 3, 2007 , and in revised form, January 18, 2008.

^* This work was supported by grants-in-aid for Scientific Research A and B from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to T. T.) and by National Institutes of Health Grants ES09558 and ES12917 (to C. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–5.

¹ To whom correspondence should be addressed: Dept. of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, B4, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Fax: 81-6-6879-3819; E-mail: todo{at}radbio.med.osaka-u.ac.jp.

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