Essential domains of Schizosaccharomyces pombe Rad8 required for DNA damage response.
Essential domains of Schizosaccharomyces pombe Rad8 required for DNA damage response.
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DOI：
10.1534/g3.114.011346
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发表时间：
2014-05-28
期刊：
影响因子：
--
通讯作者：
Forsburg SL
中科院分区：
文献类型：
--
作者：
Ding L;Forsburg SL
粟酒裂殖酵母Rad8是一个与裂殖酵母同源的保守蛋白。酿酒酵母Rad5和人HLTF，其通过促进PCNA的多泛素化而为无错误复制后修复所需。它有三个保守的结构域：E3泛素连接酶基序，SNF2家族解旋酶结构域和家族特异性HIRAN结构域。来自人类和芽殖酵母的数据表明，解旋酶活性有助于复制叉回归和模板切换叉重新启动。我们在这三个保守结构域中构建了特定的突变，发现E3连接酶和HIRAN结构域都是对各种试剂引起的DNA损伤做出适当反应所必需的。相反，解旋酶结构域中的突变在野生型背景中不显示表型。为了确定Rad8是否与其他解旋酶在功能上重叠，我们将单突变体和双突变体的表型与23种非必需解旋酶突变体的表型进行了比较，我们将其分为5个表型组。观察到突变体的rad8解旋酶影响重组的合成表型，rad8解旋酶突变影响重组突变体的一个子集的HU反应。我们的数据表明，S。粟酒属Rad8泛素连接酶活性对于响应多种破坏剂是重要的，而解旋酶结构域在特定形式的复制应激期间在调节基于重组的叉重启中仅起次要作用。
Schizosaccharomyces pombe Rad8 is a conserved protein homologous to S. cerevisiae Rad5 and human HLTF that is required for error-free postreplication repair by contributing to polyubiquitylation of PCNA. It has three conserved domains: an E3 ubiquitin ligase motif, a SNF2-family helicase domain, and a family-specific HIRAN domain. Data from humans and budding yeast suggest that helicase activity contributes to replication fork regression and template switching for fork restart. We constructed specific mutations in the three conserved domains and found that both the E3 ligase and HIRAN domains are required for proper response to DNA damage caused by a variety of agents. In contrast, mutations in the helicase domain show no phenotypes in a wild-type background. To determine whether Rad8 functionally overlaps with other helicases, we compared the phenotypes of single and double mutants with a panel of 23 nonessential helicase mutants, which we categorized into five phenotypic groups. Synthetic phenotypes with rad8∆ were observed for mutants affecting recombination, and a rad8 helicase mutation affected the HU response of a subset of recombination mutants. Our data suggest that the S. pombe Rad8 ubiquitin ligase activity is important for response to a variety of damaging agents, while the helicase domain plays only a minor role in modulating recombination-based fork restart during specific forms of replication stress.
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