Regulation of the activity of ORC.

Orc5p是构成真核生物染色体DNA复制启动子的起源识别复合体(ORC)的六个亚基之一。Orc5p包含Walker A主题。我们最近报道了一株Orc5p‘s Walker A基序(orc5-A)突变的酿酒酵母，在高温(37℃)下，细胞周期停滞在G2/M，ORC降解。ORC的另一个亚基Orc4p的过度生产特别抑制了这些表型。在这项研究中，我们研究了ORC降解的机制以及Orc4P过量生产对其抑制作用。在orc5-A中，在高温下，ORC被蛋白酶体降解；无论是添加蛋白酶体抑制剂，还是引入抑制蛋白酶体的tan1-1或nob1-4突变，都可以阻止ORC的降解。Tanl-1突变的引入恢复了细胞周期进程，表明该缺陷是由于蛋白酶体对ORC的降解所致。酵母双杂交和免疫共沉淀分析表明，Orc5p优先与Orc4p相互作用，而orc5-A突变减弱了这种相互作用。我们认为这种相互作用是由Orc4p的C-末端区域和Orc5p的N-末端区域介导的。基于这些观察，我们认为ATP与Orc5p的结合是与Orc4p有效相互作用所必需的，而在Orc5-A中，在更高的温度下失去这种相互作用使蛋白酶体降解ORC，导致生长缺陷。这个模型也可以解释为什么Orc4p的过度生产会抑制om5-A菌株的表型。

Orc5p is one of six subunits constituting the origin recognition complex (ORC), a possible initiator of chromosomal DNA replication in eukaryotes. Orc5p contains a Walker A motif. We recently reported that a strain of Saccharomyces cerevisiae having a mutation in Orc5p's Walker A motif (orc5-A), showed cell cycle arrest at G2/M and degradation of ORC at high temperatures (37°C). Over-production of Orc4p, another subunit of ORC, specifically suppressed these phenotypes. In this study, we examined the mechanisms of ORC degradation and of its suppression by Orc4p over-production. In orc5-A, at high temperatures, ORC is degraded by proteasomes; either addition of a proteasome inhibitor, or introduction of a mutation of either tanl-1 or nob1-4 that inhibits proteasomes, prevented ORC degradation. Introduction of the tanl-1 mutation restored cell cycle progression, suggesting that the defect was due to ORC degradation by proteasomes. Yeast two-hybrid and co-immunoprecipitation analyses suggested that Orc5p interacts preferentially with Orc4p and that the orc5-A mutation diminishes this interaction. We suggest that this interaction is mediated by the C-terminal region of Orc4p, and the N-terminal region of Orc5p. Based on these observations, we consider that ATP-binding to Orc5p is required for efficient interaction with Orc4p and that in orc5-A, loss of this interaction at higher temperatures allows proteasomes to degrade ORC, causing growth defects. This model could also explain why over-production of Orc4p suppresses the om5-A strain's phenotype.