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首页> 外文期刊>DNA and Cell Biology >Loss Control of Mcm5 Interaction with Chromatin in cdc6-1 Mutated in CDC-NTP Motif
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Loss Control of Mcm5 Interaction with Chromatin in cdc6-1 Mutated in CDC-NTP Motif

机译：在CDC-NTP母题突变的cdc6-1中，Mcm5与染色质相互作用的损失控制。

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Saccharomyces cerevisiae Cdc6 plays an essential role in establishing and maintaining the prereplicative complex (pre-RC) by interacting with the origin recognition complex (ORC) and associating with chromatin origins. These interactions are required to load minichromosome maintenance proteins (MCMs) and other initiator proteins onto replication origins. Although the temperature-sensitive cdc6 mutant, cdc6-1, has been widely used for these studies, the molecular mechanism of the cdc6-1 mutation has been unclear. In this study, we have identified a base substitution at Gly260 → Asp, near the CDC-NTP motif. Using a chromatin immunoprecipitation assay (CHIP), we found that cdc6-1 fails to load Mcm5 onto the replication origins. Chromatin fractions were used to study Mcm5 binding in both the wildtype and mutant background. These studies indicated that Cdc6 is also involved in unloading Mcm5 from chromatin. Specifically, the cdc6-1 mutation protein, cdc6(G260D), which failed to load Mcm5 onto replication origins, also failed to unload the Mcm5 protein. Furthermore, the overexpression of wildtype CDC6 accelerated the unloading of Mcm5 from chromatin fractions. In the absence of functional Cdc6, the Mcm5 protein showed nonorigin binding to chromatin with the cell cycle arrested at the G1S phase transition. Our results suggested that the cdc6(G260D) mutant protein fails to assemble an operational replicative complex and that wildtype Cdc6 plays a role in preventing re-replication by controlling the unloading the MCMs from chromatin origins
机译：酿酒酵母Cdc6通过与起源识别复合体（ORC）相互作用并与染色质起源相关联，在建立和维持复制前复合体（pre-RC）中起着至关重要的作用。这些相互作用是将微型染色体维持蛋白（MCM）和其他引发剂蛋白加载到复制起点上所必需的。尽管温度敏感的cdc6突变体cdc6-1已被广泛用于这些研究中，但cdc6-1突变的分子机制尚不清楚。在这项研究中，我们确定了CDC-NTP基序附近的Gly260→Asp的碱基取代。使用染色质免疫沉淀测定法（CHIP），我们发现cdc6-1无法将Mcm5加载到复制起点上。染色质级分用于研究Mcm5在野生型和突变背景中的结合。这些研究表明，Cdc6也参与了从染色质中卸载Mcm5。具体来说，无法将Mcm5加载到复制起点的cdc6-1突变蛋白cdc6（G260D）也无法卸载Mcm5蛋白质。此外，野生型CDC6的过表达加速了染色质级分中Mcm5的卸载。在缺少功能性Cdc6的情况下，Mcm5蛋白显示出与染色质的非起源结合，细胞周期停滞在G1S相变中。我们的结果表明，cdc6（G260D）突变蛋白无法组装可操作的复制复合物，而野生型Cdc6通过控制从染色质起源的MCM的卸载来防止复制。

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