Protein interaction and cellular localization of human CDC45.
Protein interaction and cellular localization of human CDC45.
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DOI：
10.1093/jb/mvt004
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发表时间：
2013-01
影响因子：
2.7
通讯作者：
Junichiro Takaya;Shunsuke Kusunoki;Y. Ishimi
中科院分区：
文献类型：
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作者：
Junichiro Takaya;Shunsuke Kusunoki;Y. Ishimi
CDC 45在真核DNA复制中起作用，是CMG（CDC 45/MCM 2 -7/GINS）复合物的成员，被认为是作为复制性DNA解旋酶发挥作用。然而，CDC 45的生物化学性质尚未完全了解。我们用免疫沉淀法系统地研究了人CDC 45与MCM 2 -7、GINS和其他复制蛋白的相互作用。我们发现，CDC 45可以直接与所有MCM 2 -7蛋白相互作用;与GINS亚基中的PSF 2，PSF 3和SLD 5相互作用;与复制蛋白A2（RPA 2），AND-1和拓扑异构酶2结合蛋白1相互作用。这些结果与CDC 45在DNA复制叉的进展中起作用的观点一致。使用针对CDC 45的抗体的实验表明，从含Triton不溶性染色质的级分中回收的CDC 45的水平在同步化的HeLa细胞中的S期中期达到峰值。然而，孵育的Triton不溶性馏分与核酸酶导致在回收率不到一半的量的CDC 45的核酸酶敏感的部分，这一结果是在与RPA 1和增殖细胞核抗原分布相反。这些结果表明，相当大一部分的CDC 45定位在一个区域以外的DNA复制叉在细胞核中，或它定位在复制叉，但它不分馏与叉蛋白，由于其紧密的联系，推测核支架。
CDC45, which plays a role in eukaryotic DNA replication, is a member of the CMG (CDC45/MCM2-7/GINS) complex that is thought to function as a replicative DNA helicase. However, the biochemical properties of CDC45 are not fully understood. We systematically examined the interactions of human CDC45 with MCM2-7, GINS and other replication proteins by immunoprecipitation. We found that CDC45 can directly interact with all MCM2-7 proteins; with PSF2, PSF3 and SLD5 in GINS subunits; and with replication protein A2 (RPA2), AND-1 and topoisomerase 2-binding protein 1. These results are consistent with the notion that CDC45 plays a role in progression of DNA replication forks. Experiments using antibodies against CDC45 show that the level of CDC45 recovered from the Triton-insoluble chromatin-containing fraction is peaked at middle of S phase in synchronized HeLa cells. However, incubation of the Triton-insoluble fraction with nucleases resulted in recovery of less than half the amount of CDC45 in the nuclease-sensitive fraction; this result is in contrast with RPA1 and proliferating cell nuclear antigen distribution. These results indicate that a considerable portion of CDC45 localizes in a region other than the DNA replication forks in nuclei or it localizes on the replication forks but it is not fractionated with the fork proteins owing to its tight association with presumably nuclear scaffolds.