Tanaka K, Yonekawa T, Kawasaki Y, Kai M, Furuya K, Iwasaki M, Murakami H, Yanagida M, Okayama H
Department of Biochemistry and Molecular Biology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Mol Cell Biol. 2000 May;20(10):3459-69. doi: 10.1128/MCB.20.10.3459-3469.2000.
Sister chromatid cohesion is essential for cell viability. We have isolated a novel temperature-sensitive lethal mutant named eso1-H17 that displays spindle assembly checkpoint-dependent mitotic delay and abnormal chromosome segregation. At the permissive temperature, the eso1-H17 mutant shows mild sensitivity to UV irradiation and DNA-damaging chemicals. At the nonpermissive temperature, the mutant is arrested in M phase with a viability loss due to a failure to establish sister chromatid cohesion during S phase. The lethal M-phase arrest phenotype, however, is suppressed by inactivation of a spindle checkpoint. The eso1(+) gene is not essential for the onset and progression of DNA replication but has remarkable genetic interactions with those genes regulating the G(1)-S transition and DNA replication. The N-terminal two-thirds of Eso1p is highly homologous to DNA polymerase eta of budding yeast and humans, and the C-terminal one-third is homologous to budding yeast Eco1p (also called Ctf7p), which is required for the establishment of sister chromatid cohesion. Deletion analysis and determination of the mutation site reveal that the function of the Eco1p/Ctf7p-homologous domain is necessary and sufficient for sister chromatid cohesion. On the other hand, deletion of the DNA polymerase eta domain in Eso1p increases sensitivity to UV irradiation. These results indicate that Eso1p plays a dual role during DNA replication. The C-terminal region acts to establish sister chromatid cohesion, and the N-terminal region presumably catalyzes translesion DNA synthesis when template DNA contains lesions that block regular DNA replication.
姐妹染色单体黏连对于细胞存活至关重要。我们分离出了一个名为eso1-H17的新型温度敏感致死突变体,它表现出纺锤体组装检查点依赖性的有丝分裂延迟和异常的染色体分离。在允许温度下,eso1-H17突变体对紫外线照射和DNA损伤化学物质表现出轻度敏感性。在非允许温度下,该突变体在M期停滞,由于在S期未能建立姐妹染色单体黏连而导致活力丧失。然而,纺锤体检查点的失活可抑制致死性的M期停滞表型。eso1(+)基因对于DNA复制的起始和进程并非必需,但与那些调节G(1)-S转变和DNA复制的基因具有显著的遗传相互作用。Eso1p的N端三分之二与芽殖酵母和人类的DNA聚合酶η高度同源,而C端三分之一与芽殖酵母Eco1p(也称为Ctf7p)同源,后者是建立姐妹染色单体黏连所必需的。缺失分析和突变位点的确定表明,Eco1p/Ctf7p同源结构域的功能对于姐妹染色单体黏连是必要且充分的。另一方面,Eso1p中DNA聚合酶η结构域的缺失增加了对紫外线照射的敏感性。这些结果表明,Eso1p在DNA复制过程中发挥双重作用。C端区域负责建立姐妹染色单体黏连,而N端区域可能在模板DNA含有阻碍正常DNA复制的损伤时催化跨损伤DNA合成。