Chavdarova Melita, Marini Victoria, Sisakova Alexandra, Sedlackova Hana, Vigasova Dana, Brill Steven J, Lisby Michael, Krejci Lumir
Department of Biology, Masaryk University, Kamenice 5/A7, Brno 625 00, Czech Republic National Centre for Biomolecular Research, Masaryk University, Kamenice 5/A4, Brno 625 00, Czech Republic.
Department of Biology, Masaryk University, Kamenice 5/A7, Brno 625 00, Czech Republic.
Nucleic Acids Res. 2015 Apr 20;43(7):3626-42. doi: 10.1093/nar/gkv198. Epub 2015 Mar 12.
A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81-Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81-Mms4. In this study, we show that the Srs2 and Mus81-Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81-Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81-Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81-Mms4 to cleave DNA. Concomitantly, Mus81-Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81-Mms4 and Srs2 in processing of recombination as well as replication intermediates.
DNA模板内的多种DNA损伤、二级DNA结构或拓扑应力可能导致复制叉停滞。恢复此类复制叉对于维持基因组稳定性至关重要。结构特异性核酸内切酶Mus81-Mms4参与处理因复制叉坍塌和同源重组产生的DNA中间体。根据先前的遗传学研究，Srs2解旋酶可能与Mus81-Mms4一起在双链断裂和单链DNA缺口的修复中发挥作用。在本研究中，我们表明Srs2和Mus81-Mms4蛋白在体外和体内发生物理相互作用，并且我们绘制了Srs2和Mus81蛋白内的相互作用结构域。此外，我们表明Srs2在刺激Mus81-Mms4对多种DNA底物的核酸酶活性方面发挥双重作用。首先，Srs2直接刺激Mus81-Mms4核酸酶活性，而与其解旋酶活性无关。其次，Srs2从DNA上移除Rad51，以使Mus81-Mms4能够接近并切割DNA。同时，Mus81-Mms4抑制Srs2的解旋酶活性。综上所述，我们的数据表明Mus81-Mms4和Srs2在处理重组以及复制中间体方面具有协同作用。
