Human Werner helicase interacting protein 1 (WRNIP1) functions as a novel modulator for DNA polymerase δ
Human Werner helicase interacting protein 1 (WRNIP1) functions as a novel modulator for DNA polymerase δ
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DOI：
10.1111/j.1365-2443.2004.00812.x
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发表时间：
2004-12
期刊：
影响因子：
2.1
通讯作者：
T. Tsurimoto;Ayako Shinozaki;M. Yano;M. Seki;T. Enomoto
中科院分区：
文献类型：
--
作者：
T. Tsurimoto;Ayako Shinozaki;M. Yano;M. Seki;T. Enomoto
人类 WRNIP1 是一种 Werner DNA 解旋酶相互作用蛋白 1，在昆虫细胞和大肠杆菌中表达。纯化的蛋白质表现为同源寡聚复合物，其天然分子量表示八聚体，并且该复合物与受双链 DNA 末端刺激的 ATP 酶活性共纯化。根据芽殖酵母 WRNIP1/Mgs1 的遗传学研究表明，纯化的人 WRNIP1 复合物与人 DNA 聚合酶 δ (pol δ) 发生物理相互作用，在存在或不存在增殖细胞核抗原的情况下，将其 DNA 合成活性刺激五倍以上。反应产物的分析表明，刺激部分是由于 pol δ 的持续合成能力增加，但更重要的是其引发频率的增加。向反应中添加 ATP 部分抑制了 WRNIP1 的刺激。此外，缺乏 ATPase 活性的突变体 WRNIP1 可以正常刺激 pol δ，但对 ATP 的抑制不敏感。这些结果表明，WRNIP1 在 pol δ 介导的 DNA 合成过程中充当启动或重启事件的调节剂，并且其 ATP 酶活性用于感知 DNA 末端并调节刺激程度。
Human WRNIP1, a Werner DNA helicase interacting protein 1, was expressed in insect cells and E. coli. The purified protein behaved as a homo‐oligomeric complex with a native molecular mass indicative of an octamer, and the complex copurified with an ATPase activity that was stimulated by double‐stranded DNA ends. As suggested by genetic studies of budding yeast WRNIP1/Mgs1, the purified human WRNIP1 complex interacted physically with human DNA polymerase δ (pol δ), stimulating its DNA synthesis activity more than fivefold in the presence or absence of proliferating cell nuclear antigen. Analysis of reaction products demonstrated the stimulation to be partly due to an increased processivity of pol δ but more importantly to an increase in its initiation frequency. Addition of ATP to reactions partially suppressed stimulation by WRNIP1. Furthermore, a mutant WRNIP1 lacking ATPase activity could stimulate pol δ normally but was insensitive to suppression by ATP. These results indicate that WRNIP1 functions as a modulator for initiation or restart events during pol δ‐mediated DNA synthesis and that its ATPase activity is utilized to sense DNA ends and to regulate the extent of stimulation.