Kinetics of ATP Binding to the Origin Recognition Complex of Saccharomyces cerevisiae

Masaki Makise, Hitomi Takenaka, Wakako Kuwae, Naoko Takahashi, Tomofusa Tsuchiya, Tohru Mizushima

研究成果: Article査読

14 被引用数 (Scopus)

抄録

Origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, binds specifically to ATP through two of its subunits (Orc1p and Orc5p). In this study, we investigated the kinetics of ATP binding to ORC by a filter binding assay. The Kd values for the ATP of wild-type ORC and ORC-1A (mutant ORC containing Orc1p with a defective Walker A motif) were less than 10 nM, suggesting that the affinity of Orc5p for ATP is very high. On the other hand, the Kd values for the ATP of ORC-5A (mutant ORC containing Orc5p with a defective Walker A motif) was much higher (about 1.5 μM), suggesting that the affinity of Orc1p for ATP is relatively low in the absence of origin DNA. ATP dissociated more rapidly from its complex with ORC-5A than from its complex with ORC-1A, suggesting that the ATP-Orc5p complex is more stable than ATP-Orc1p complex. Origin DNA fragments decreased the Kd value of ORC-5A for ATP and stabilized the complex of ATP with ORC-5A. Wild-type ORC, ORC-1A, and ORC-5A required different concentrations of ATP for specific binding to origin DNA. All of these results imply that ATP binding to Orc5p, ATP binding to Orc1p, and origin DNA binding to ORC are co-operatively regulated, which may be important for the initiation of DNA replication.

本文言語English
ページ(範囲)46440-46445
ページ数6
ジャーナルJournal of Biological Chemistry
278
47
DOI
出版ステータスPublished - 2003 11月 21

ASJC Scopus subject areas

  • 生化学
  • 分子生物学
  • 細胞生物学

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