MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts
MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts
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DOI：
10.1074/jbc.m204438200
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发表时间：
2002-09-06
影响因子：
4.8
通讯作者：
Walter, JC
中科院分区：
文献类型：
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作者：
Edwards, MC;Tutter, AV;Walter, JC
MCM2-7复合体被认为是真核复制的DNA解旋酶。它被起源识别复合体、CdC6和CdT1募集到染色质中，并在G(1)/S转换时被CdC45和蛋白激酶CdC7和Cdk2激活。矛盾的是，与染色质结合的MCM复合体的数量远远超过结合的ORC复合体的数量。为了了解MCM2-7：ORC的高比率是如何产生的，我们检测了这些蛋白质与非洲爪哇卵提取液中固定的线性DNA片段的结合。招募ORC和MCM2-7所需的DNA最小长度类似于80bp，该片段上的MCM2-7：ORC比率类似于1：1。随着DNA片段的增加，MCM2-7：ORC比率显著增加，这表明MCM复合体通常分布在ORC周围的DNA大范围内。在DNA复制开始时，只有一小部分染色质结合的MCM2-7复合体招募了CDC45，并且与CDC45不同，MCM2-7对DNA复制没有限制。然而，所有染色质结合的MCM复合体都可能是功能性的，因为它们是以依赖于CDC7的方式被磷酸化的，并且因为它们可以被诱导来支持依赖于CDK2的CDC45负载。这些数据表明，在非洲爪蛙卵提取液中，复制起源包含多个分布的、具有启动能力的MCM2-7复合体。
The MCM2-7 complex is believed to function as the eukaryotic replicative DNA helicase. It is recruited to chromatin by the origin recognition complex (ORC), Cdc6, and Cdt1, and it is activated at the G(1)/S transition by Cdc45 and the protein kinases Cdc7 and Cdk2. Paradoxically, the number of chromatin-bound MCM complexes greatly exceeds the number of bound ORC complexes. To understand how the high MCM2-7:ORC ratio comes about, we examined the binding of these proteins to immobilized linear DNA fragments in Xenopus egg extracts. The minimum length of DNA required to recruit ORC and MCM2-7 was similar to80 bp, and the MCM2-7: ORC ratio on this fragment was similar to1:1. With longer DNA fragments, the MCM2-7:ORC ratio increased dramatically, indicating that MCM complexes normally become distributed over a large region of DNA surrounding ORC. Only a small subset of the chromatin-bound MCM2-7 complexes recruited Cdc45 at the onset of DNA replication, and unlike Cdc45, MCM2-7 was not limiting for DNA replication. However, all the chromatin-bound MCM complexes may be functional, because they were phosphorylated in a Cdc7-dependent fashion, and because they could be induced to support Cdk2-dependent Cdc45 loading. The data suggest that in Xenopus egg extracts, origins of replication contain multiple, distributed, initiation-competent MCM2-7 complexes.