Maestroni Laetitia, Audry Julien, Luciano Pierre, Coulon Stéphane, Géli Vincent, Corda Yves
Aix-Marseille Univ, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, Marseille, France. Equipe Labellisée par la Ligue Nationale contre le Cancer.
Cell Stress. 2020 Jan 17;4(3):48-63. doi: 10.15698/cst2020.03.214.
In , 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 mutation that lowers the affinity of RPA in particular for G-rich single-stranded DNA. At the lagging strand, in contrast to Δ, the 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 mutant indicating that Pif1 can also act at the lagging strand. We show that the effects of the ( in fission yeast) are conserved in . Finally, we report that RNase H1 interacts in a DNA-dependent manner with RPA in budding yeast, however overexpression of RNase H1 does not rescue CEB1 instability observed in Δ and 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.
在……中，Pif1解旋酶的缺失会导致在前导链而非后随链复制过程中含G4的CEB1微卫星的不稳定性。我们报告称，当形成G4的链位于前导链或后随链模板上时，RPA和Pif1协同作用以维持CEB1的稳定性。在前导链上，RPA与Pif1作用于相同途径以维持CEB1的稳定性。与该结果一致，RPA与Pif1共沉淀。Pif1与RPA之间的这种关联受到……突变的影响，该突变降低了RPA对富含G的单链DNA的亲和力。在后随链上，与Δ……相反，……突变强烈增加了CEB1重排的频率。我们解释说，在后随链DNA上Pif1是可有可无的，因为RPA自身有能力在后随链合成过程中防止形成稳定的富含G的二级结构。值得注意的是，Pif1的过表达挽救了……突变体中后随链上CEB1的不稳定性，表明Pif1也能在后随链上发挥作用。我们表明……（裂殖酵母中的……）的效应在……中是保守的。最后，我们报告称，在芽殖酵母中RNase H1以DNA依赖的方式与RPA相互作用，然而RNase H1的过表达并不能挽救在Δ……和……突变体中观察到的CEB1不稳定性。这些结果共同为RPA在防止DNA二级结构形成以及协调旨在解决这些结构的因子作用方面的一般作用提供了新的见解。
