The alternative Ctf18-Dcc1-Ctf8-replication factor C complex required for sister chromatid cohesion loads proliferating cell nuclear antigen onto DNA
The alternative Ctf18-Dcc1-Ctf8-replication factor C complex required for sister chromatid cohesion loads proliferating cell nuclear antigen onto DNA
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DOI：
10.1073/pnas.1434308100
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发表时间：
2003-09-02
影响因子：
11.1
通讯作者：
Hurwitz, J
中科院分区：
文献类型：
--
作者：
Bermudez, VP;Maniwa, Y;Hurwitz, J
DNA复制后姐妹染色单体的连接确保了子细胞对染色体的忠实继承。在芽殖酵母中，姐妹染色单体内聚力的建立需要 Ctf8、Dcc1 和 Ctf18（复制因子 C (RFC) 的 p140 亚基的同源物）。在本报告中，我们证明在 293T 细胞中，Flag 标记的 Ctf18 形成由 Ctf18、Dcc1、Ctf8、RFCp40、RFCp38、RFCp37 和 RFCp36 (Ctf 18-RFC) 组成的七亚基内聚-RFC 复合物。我们证明，可以通过在杆状病毒感染的昆虫细胞中共表达七种蛋白质来组装化学计量的异七聚 Ctf 18-RFC 复合物。此外，还形成了另外两个稳定的子复合物，其中包括由Ctf18、RFCp40、RFCp38、RFCp37和RFCp36组成的五聚体复合物和二聚体Dcc1-Ctf8。五亚基和七亚基 Ctf18-RFC 复合物均与单链和引发的 DNA 结合，并具有微弱的 ATP 酶活性，该活性可通过添加引发的 DNA 和增殖细胞核抗原 (PCNA) 来刺激。这些复合物催化 PCNA 依赖于 ATP 加载到引物和缺口 DNA 上，但不催化到双链切口或单链环状 DNA 上。与这些观察结果一致，在 PCNA 依赖性 DNA 聚合酶 δ 催化的 DNA 复制反应中，两种 Ctf18-RFC 复合物都取代了复制性 RFC。这些结果支持姐妹染色单体内聚力与 DNA 复制相关的模型。
The linkage of sister chromatids after DNA replication ensures the faithful inheritance of chromosomes by daughter cells. In budding yeast, the establishment of sister chromatid cohesion requires Ctf8, Dcc1, and Ctf18, a homologue of the p140 subunit of the replication factor C (RFC). In this report we demonstrate that in 293T cells, Flag-tagged Ctf18 forms a seven-subunit cohesion-RFC complex comprised of Ctf18, Dcc1, Ctf8, RFCp40, RFCp38, RFCp37, and RFCp36 (Ctf 18-RFC). We demonstrate that a stoichiometric heteroheptameric Ctf 18-RFC complex can be assembled by coexpressing the seven proteins in baculovirus-infected insect cells. In addition, the two other stable subcomplexes were formed, which include a pentameric complex comprised of Ctf18, RFCp40, RFCp38, RFCp37, and RFCp36 and a dimeric Dcc1-Ctf8. Both the five- and seven-subunit Ctf18-RFC complexes bind to single-stranded and primed DNAs and possess weak ATPase activity that is stimulated by the addition of primed DNA and proliferating cell nuclear antigen (PCNA). These complexes catalyzed the ATP-dependent loading of PCNA onto primed and gapped DNA but not onto double-stranded nicked or single-stranded circular DNAs. Consistent with these observations, both Ctf18-RFC complexes substituted for the replicative RFC in the PCNA-dependent DNA polymerase delta-catalyzed DNA replication reaction. These results support a model in which sister chromatid cohesion is linked to DNA replication.