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J. Biol. Chem., Vol. 282, Issue 29, 20906-20914, July 20, 2007

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A Second Proliferating Cell Nuclear Antigen Loader Complex, Ctf18-Replication Factor C, Stimulates DNA Polymerase Activity^*

Yasushi Shiomi, Chikahide Masutani, Fumio Hanaoka, Hiroshi Kimura^¶, and Toshiki Tsurimoto¹

From the Department of Biology, School of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan, the Graduate School of Frontier Biosciences, Osaka University and CREST, Japan Science and Technology Corp., 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan, and ^¶Horizontal Medical Research Organization, School of Medicine, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

Replication factor C (RFC) loads the clamp protein PCNA onto DNA structures. Ctf18-RFC, which consists of the chromosome cohesion factors Ctf18, Dcc1, and Ctf8 and four small RFC subunits, functions as a second proliferating cell nuclear antigen (PCNA) loader. To identify potential targets of Ctf18-RFC, human cell extracts were assayed for DNA polymerase activity specifically stimulated by Ctf18-RFC in conjunction with PCNA. After several chromatography steps, an activity stimulated by Ctf18-RFC but not by RFC was identified. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis revealed the presence of two DNA polymerases, and , in the most purified fraction, but experiments with purified recombinant proteins demonstrated that only polymerase (pol) was responsible for activity. Ctf18-RFC alone stimulated pol , and the addition of PCNA cooperatively increased stimulation. Furthermore, Ctf18-RFC interacted physically with pol , as indicated by co-precipitation in human cells. We propose that this novel loader-DNA polymerase interaction allows DNA replication forks to overcome interference by various template structures, including damaged DNA and DNA-protein complexes that maintain chromosome cohesion.

Received for publication, October 30, 2006 , and in revised form, May 9, 2007.

^* This work was supported by grants-in-aid for scientific research (B) and scientific research on priority areas from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. 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. S1 and S2.

¹ To whom correspondence should be addressed. Tel.: 81-92-642-2613; Fax: 81-92-642-2645; E-mail: ttsurscb{at}mbox.nc.kyushu-u.ac.jp.

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