Yeast two-hybrid analysis of the origin recognition complex of Saccharomyces cerevisiae:: interaction between subunits and identification of binding proteins
Yeast two-hybrid analysis of the origin recognition complex of Saccharomyces cerevisiae:: interaction between subunits and identification of binding proteins
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DOI：
10.1111/j.1567-1364.2007.00298.x
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发表时间：
2007-12-01
影响因子：
3.2
通讯作者：
Mizushima, Tohru
中科院分区：
文献类型：
--
作者：
Matsuda, Kazuya;Makise, Masaki;Mizushima, Tohru
起源识别复合物（originrecognitioncomplex，ORC）是一种由六个蛋白质组成的复合物，是真核生物中最有可能启动染色体DNA复制的蛋白质。虽然从生物化学和遗传学角度对酿酒酵母的ORC进行了广泛的研究，但其四级结构仍然未知。以前的研究表明，ORC具有DNA复制以外的功能，如基因沉默，但这些功能的分子机制尚未确定。在本研究中，我们使用酵母双杂交分析来研究ORC亚基之间的相互作用，并寻找ORC结合蛋白。除了已知的Orc 4p-Orc 5 p相互作用外，我们还发现Orc 2 p和Ord 3 p（2 p-3 p）、Orc 2 p和Ord 5 p（2 p-5 p）、Orc 2 p和Ord 6p（2 p-6p）以及Orc 3 p和Ord 6p（3 p-6p）之间存在强相互作用，而Orc 1 p和Ord 4p（1 p-4p）、Orc 3 p和Ord 4p（3 p-4p）之间存在弱相互作用，Orc 2 p和Ord 3 p（3 p-5 p）以及Orc 5 p和Ord 3 p（5 p-6p）。这些结果表明，2 p-3 p-6p可能形成一个核心复合物。Orc 2 p和Orc 6p在体内被磷酸化，调节DNA复制的起始。然而，用不能被磷酸化或模拟磷酸化的其他氨基酸残基替换磷酸化的氨基酸残基并不影响亚基相互作用。我们还鉴定了几种与ORC亚基相互作用的蛋白质; Sir 4p和Mad 1 p与Orc 2 p相互作用; Cac 1 p和Ykr 077 wp与Orc 3 p相互作用; Rrm 3 p和Swi 6p与Orc 5 p相互作用; Mih 1 p与Orc 6p相互作用。我们讨论了这些相互作用在ORC功能中的作用。
Origin recognition complex (ORC), a six-protein complex (Orc1p-6p), is the most likely initiator of chromosomal DNA replication in eukaryotes. Although ORC of Saccharomyces cerevisiae has been studied extensively from biochemical and genetic perspectives, its quaternary structure remains unknown. Previous studies suggested that ORC has functions other than DNA replication, such as gene silencing, but the molecular mechanisms of these functions have not been determined. In this study, we used yeast two-hybrid analysis to examine the interaction between ORC subunits and to search for ORC-binding proteins. As well as the known Orc4p-Orc5p interaction, we revealed strong interactions between Orc2p and Ord3p (2p-3p), Orc2p and Ord5p (2p-5p), Orc2p and Ord6p (2p-6p) and Orc3p and Ord6p (3p-6p) and weaker interactions between Orc1p and Ord4p (1p-4p), Orc3p and Ord4p (3p-4p), Orc2p and Ord3p (3p-5p) and Orc5p and Ord3p (5p-6p). These results suggest that 2p-3p-6p may form a core complex. Orc2p and Orc6p are phosphorylated in vivo, regulating initiation of DNA replication. However, replacing the phosphorylated amino acid residues with others that cannot be phosphorylated, or that mimic phosphorylation, did not affect subunit interactions. We also identified several proteins that interact with ORC subunits; Sir4p and Mad1p interact with Orc2p; Cac1p and Ykr077wp with Orc3p; Rrm3p and Swi6p with Orc5p; and Mih1p with Orc6p. We discuss roles of these interactions in functions of ORC.