Ding Lin, Forsburg Susan L
Program in Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089-2910.
Program in Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089-2910
G3 (Bethesda). 2014 May 28;4(8):1373-84. doi: 10.1534/g3.114.011346.
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.
粟酒裂殖酵母Rad8是一种与酿酒酵母Rad5和人类HLTF同源的保守蛋白，它通过促进增殖细胞核抗原（PCNA）的多聚泛素化参与无差错的复制后修复。它有三个保守结构域：一个E3泛素连接酶基序、一个SNF2家族解旋酶结构域和一个家族特异性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的回退和用于叉重新启动的模板切换。我们在这三个保守结构域中构建了特定突变，发现E3连接酶结构域和HIRAN结构域对于正确应对由多种试剂引起的DNA损伤都是必需的。相比之下，解旋酶结构域的突变在野生型背景下没有表型。为了确定Rad8是否在功能上与其他解旋酶重叠，我们将单突变体和双突变体的表型与一组23个非必需解旋酶突变体进行了比较，我们将这些突变体分为五个表型组。对于影响重组的突变体，观察到了与rad8∆的合成表型，并且rad8解旋酶突变影响了一部分重组突变体对羟基脲（HU）的反应。我们的数据表明，粟酒裂殖酵母Rad8泛素连接酶活性对于应对多种损伤试剂很重要，而解旋酶结构域在特定形式的复制应激期间调节基于重组的叉重新启动中仅起次要作用。
