在非洲爪蟾卵提取物中,MCM2 - 7复合物以围绕起始识别复合物的分散模式结合染色质。
MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts.
作者信息
Edwards Melissa C, Tutter Antonin V, Cvetic Christin, Gilbert Catherine H, Prokhorova Tatyana A, Walter Johannes C
机构信息
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
出版信息
J Biol Chem. 2002 Sep 6;277(36):33049-57. doi: 10.1074/jbc.M204438200. Epub 2002 Jun 26.
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 approximately 80 bp, and the MCM2-7:ORC ratio on this fragment was approximately 1:1. 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.
MCM2 - 7复合物被认为作为真核生物复制性DNA解旋酶发挥作用。它通过起始识别复合物(ORC)、Cdc6和Cdt1被招募到染色质上,并在G(1)/S期转换时被Cdc45以及蛋白激酶Cdc7和Cdk2激活。矛盾的是,与染色质结合的MCM复合物的数量大大超过结合的ORC复合物的数量。为了理解高MCM2 - 7:ORC比例是如何形成的,我们在非洲爪蟾卵提取物中研究了这些蛋白质与固定化线性DNA片段的结合情况。招募ORC和MCM2 - 7所需的DNA最短长度约为80 bp,并且该片段上的MCM2 - 7:ORC比例约为1:1。对于更长的DNA片段,MCM2 - 7:ORC比例急剧增加,这表明MCM复合物通常分布在围绕ORC的大片段DNA区域上。在DNA复制开始时,只有一小部分与染色质结合的MCM2 - 7复合物招募了Cdc45,并且与Cdc45不同,MCM2 - 7对DNA复制并非限制性因素。然而,所有与染色质结合的MCM复合物可能都具有功能,因为它们以依赖Cdc7的方式被磷酸化,并且因为它们可以被诱导支持依赖Cdk2的Cdc45加载。这些数据表明,在非洲爪蟾卵提取物中,复制起点包含多个分布的、具有起始能力的MCM2 - 7复合物。