Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks
Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks
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DOI：
10.1128/mcb.00471-09
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发表时间：
2009-09-01
影响因子：
5.3
通讯作者：
Whitby, Matthew C.
中科院分区：
文献类型：
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作者：
Lorenz, Alexander;Osman, Fekret;Whitby, Matthew C.
控制Rad51在DNA上的负载对于控制何时以及如何使用同源重组是很重要的。在这里，我们使用遗传分析和间接免疫荧光相结合的方法来证明F-box DNA解旋酶（Fbh1）的功能与Rad52同源物Rad22直接相反，以抑制Rad51装载到裂变酵母的DNA上。令人惊讶的是，这种活性对于限制自发的直接重复重组是不必要的。相反，当复制分叉被阻塞和/或破坏时，它似乎在防止重组方面发挥了重要作用。当过表达时，Fbh1特异性地减少复制叉块诱导的重组，以及由复制应激诱导的Rad51核病灶的数量。这些能力依赖于它的DNA解旋酶/转位酶活性，这表明Fbh1通过作为Rad51破坏酶来控制重组。与此一致的是，在缺乏Rad51破坏酶Srs2的菌株中，Fbh1的过表达也抑制了重组蛋白的高水平形成和Rad51在位点特异性复制叉屏障上的积累。类似地，在fbh1 Delta突变体中，Srs2的过表达抑制了复制叉块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，不易被Srs2取代。
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 fbh1 Delta mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.