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J. Biol. Chem., Vol. 279, Issue 9, 8469-8477, February 27, 2004

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Analysis on Origin Recognition Complex containing Orc5p with defective Walker A Motif^*

Naoko Takahashi, Yoshihiro Yamaguchi, Fumiko Yamairi, Masaki Makise, Hitomi Takenaka, Tomofusa Tsuchiya, and Tohru Mizushima

From the Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan

Orc5p is one of six proteins that make up the origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes. To investigate the role of ATP binding to Orc5p in cells, we constructed orc5-A, a strain of Saccharomyces cerevisiae having a mutation in the Walker A motif of Orc5p (K43E). The strain showed temperature-sensitive growth. Incubation at a nonpermissive temperature (37 °C) caused accumulation of cells with nearly 2C DNA content. Overproduction of Orc4p, another subunit of ORC, suppresses this temperature sensitivity, but overproduction of other subunits did not. Overproduction of Orc4p did not suppress the temperature sensitivity of another orc5 mutant, orc5-1, whose mutation, L331P, is outside the ATP-binding motif. These results suggest that Orc4p is specifically involved in ATP binding to Orc5p itself or its function in DNA replication. Immunoblotting experiments revealed that in the orc5-A strain at a nonpermissive temperature, all ORC subunits gradually disappeared, suggesting that ORC5-A becomes degraded at nonpermissive temperatures. We therefore consider that ATP binding to Orc5p is involved in efficient ORC formation and that Orc4p is involved in this process.

Received for publication, May 27, 2003 , and in revised form, November 17, 2003.

^* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology (Japan), by the Asahi Glass Foundation, by the Naito Foundation, and by the Kato Memorial Foundation. 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.

Research Fellow of the Japan Society for the Promotion of Science.

To whom correspondence should be addressed: Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Okayama 700-8530, Japan. Tel. and Fax: 81-86-251-7958; E-mail: mizushima{at}pharm.okayama-u.ac.jp.

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