RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand
RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand
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DOI：
10.15698/cst2020.03.214
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发表时间：
2020-03-01
期刊：
影响因子：
6.4
通讯作者：
Corda, Yves
中科院分区：
文献类型：
--
作者：
Maestroni, Laetitia;Audry, Julien;Corda, Yves
在酿酒酵母中，Pif 1解旋酶的缺乏诱导的不稳定性的G4-含有CEB 1小卫星在前导链，但不是滞后链复制。我们报告说，RPA和Pif 1合作，以维持CEB 1的稳定性时，G4形成链的领先或落后的链模板。在前导链，RPA与Pif 1以相同的途径起作用，以维持CEB 1的稳定性。与该结果一致，RPA与Pif 1共沉淀。Pif 1和RPA之间的这种关联受到rfa 1-D228 Y突变的影响，该突变降低了RPA的亲和力，特别是对富含G的单链DNA的亲和力。在滞后链，与pif 11相反，rfa 1-D228 Y突变强烈增加CEB 1重排的频率。我们解释说，Pif 1是通过RPA本身的能力，以防止形成稳定的G-丰富的二级结构，在滞后链合成的滞后链DNA。值得注意的是，Pif 1的过表达挽救了rfa 1-D228 Y突变体中CEB 1在滞后链处的不稳定性，表明Pif 1也可以作用于滞后链。我们发现rfa 1-D228 Y（裂殖酵母中的rpal-D223 Y）的作用在粟酒裂殖酵母中是保守的。最后，我们报告说，RNase H1相互作用的DNA依赖性的方式与RPA在芽殖酵母，然而，RNase H1的过表达不能挽救CEB 1的不稳定性观察pif 1 Delta和rfa 1-D228 Y突变体。总的来说，这些结果增加了关于RPA在防止DNA二级结构形成和协调旨在解决它们的因素的作用方面的一般作用的新见解。
In Saccharomyces cerevisiae, the absence of Pif1 helicase induces the instability of G4-containing CEB1 minisatellite during leading strand but not lagging strand replication. We report that RPA and Pif1 cooperate to maintain CEB1 stability when the G4 forming strand is either on the leading or lagging strand templates. At the leading strand, RPA acts in the same pathway as Pif1 to maintain CEB1 stability. Consistent with this result, RPA co-precipitates with Pif1. This association between Pif1 and RPA is affected by the rfa1-D228Y mutation that lowers the affinity of RPA in particular for G-rich single-stranded DNA. At the lagging strand, in contrast to pif11, the rfa1-D228Y mutation strongly increases the frequency of CEB1 rearrangements. We explain that Pif1 is dispensable at the lagging strand DNA by the ability of RPA by itself to prevent formation of stable G-rich secondary structures during lagging strand synthesis. Remarkably, overexpression of Pif1 rescues the instability of CEB1 at the lagging strand in the rfa1-D228Y mutant indicating that Pif1 can also act at the lagging strand. We show that the effects of the rfa1-D228Y (rpal-D223Y in fission yeast) are conserved in Schizosaccharomyces pombe. Finally, we report that RNase H1 interacts in a DNAdependent manner with RPA in budding yeast, however overexpression of RNase H1 does not rescue CEB1 instability observed in pif1 Delta and rfa1-D228Y mutants. Collectively these results add new insights about the general role of RPA in preventing formation of DNA secondary structures and in coordinating the action of factors aimed at resolving them.