Lee Jong-Ryul, Makise Masaki, Takenaka Hitomi, Takahashi Naoko, Yamaguchi Yoshihiro, Tsuchiya Tomofusa, Mizushima Tohru
Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Okayama 700-8530, Japan.
Biochem J. 2002 Mar 1;362(Pt 2):395-9. doi: 10.1042/bj3620395.
Origin-recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, shares certain biochemical characteristics with DnaA, the initiator of chromosomal DNA replication in prokaryotes. These similarities include origin-specific DNA binding, ATP binding and ATPase activity. DnaA interacts with acidic phospholipids, such as cardiolipin, and its activity is regulated by these phospholipids. In this study, we examined whether Saccharomyces cerevisiae ORC also interacts with phospholipids. Among the various phospholipids tested, ORC was found to bind specifically to cardiolipin. This binding was inhibited by excess concentrations of salts but unaffected by ATP, adenosine 5'-[gamma-thio]triphosphate or the origin DNA. Cardiolipin weakly inhibited the ATP-binding activity of ORC, whereas it strongly inhibited ORC binding to origin DNA. Acidic phospholipids other than cardiolipin (phosphatidylglycerol and phosphatidylinositol) weakly inhibited ORC binding to origin DNA. Furthermore, total phospholipids extracted from yeast nuclear membranes inhibited ORC binding to origin DNA. We consider that phospholipids may modulate initiation of DNA replication in eukaryotes in a similar manner to that found in prokaryotes.
起始识别复合物（ORC）是真核生物中染色体DNA复制的候选起始因子，它与原核生物中染色体DNA复制的起始因子DnaA具有某些生化特性。这些相似之处包括对特定起始位点的DNA结合、ATP结合和ATP酶活性。DnaA与酸性磷脂（如心磷脂）相互作用，其活性受这些磷脂调节。在本研究中，我们检测了酿酒酵母ORC是否也与磷脂相互作用。在测试的各种磷脂中，发现ORC特异性结合心磷脂。这种结合受到过量盐浓度的抑制，但不受ATP、腺苷5'-[γ-硫代]三磷酸或起始位点DNA的影响。心磷脂对ORC的ATP结合活性有微弱抑制作用，而对ORC与起始位点DNA的结合有强烈抑制作用。除心磷脂外的其他酸性磷脂（磷脂酰甘油和磷脂酰肌醇）对ORC与起始位点DNA的结合有微弱抑制作用。此外，从酵母核膜中提取的总磷脂抑制ORC与起始位点DNA的结合。我们认为磷脂可能以与原核生物中类似的方式调节真核生物中的DNA复制起始。
