Lorenz Alexander, Osman Fekret, Folkyte Victoria, Sofueva Sevil, Whitby Matthew C
Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
Mol Cell Biol. 2009 Sep;29(17):4742-56. doi: 10.1128/MCB.00471-09. Epub 2009 Jun 22.
Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Delta mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.
控制Rad51在DNA上的装载对于调控同源重组的使用时间和方式至关重要。在此，我们结合遗传分析和间接免疫荧光实验表明，F-box DNA解旋酶（Fbh1）的功能与Rad52直系同源物Rad22直接相反，可抑制裂殖酵母中Rad51在DNA上的装载。令人惊讶的是，这种活性对于限制自发的直接重复重组并非必需。相反，它似乎在复制叉受阻和/或断裂时防止重组中发挥重要作用。当Fbh1过表达时，它会特异性降低复制叉阻断诱导的重组以及复制应激诱导的Rad51核灶数量。这些能力依赖于其DNA解旋酶/转位酶活性，表明Fbh1通过作为Rad51解聚酶来对重组施加控制。与此一致的是，在缺乏Rad51解聚酶Srs2的菌株中，Fbh1的过表达也抑制了在位点特异性复制叉屏障处高水平的重组形成和Rad51积累。同样，Srs2的过表达抑制了fbh1Δ突变体中复制叉阻断诱导的基因转换事件，尽管无法抑制缺失事件表明Fbh1具有独特的功能，而Srs2不易替代该功能。
