RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand.

IF 4.1 Q2 CELL BIOLOGY
Laetitia Maestroni, Julien Audry, Pierre Luciano, Stéphane Coulon, Vincent Géli, Yves Corda
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引用次数: 22

Abstract

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 pif1Δ, 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 (rpa1-D223Y in fission yeast) are conserved in Schizosaccharomyces pombe. 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 pif1Δ 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.

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RPA和Pif1共同去除前链和后链上的富g结构。
在酿酒酵母中,Pif1解旋酶的缺失诱导含有g4的CEB1小卫星在前导链复制过程中不稳定,但在后导链复制过程中不稳定。我们报道,当G4形成链位于前导链或滞后链模板上时,RPA和Pif1合作维持CEB1的稳定性。在前导链上,RPA与Pif1通过相同的途径维持CEB1的稳定性。与此结果一致,RPA与Pif1共析出。Pif1和RPA之间的这种关联受到rfa1-D228Y突变的影响,该突变降低了RPA特别是对富含g的单链DNA的亲和力。在后链上,与pif1Δ相反,rfa1-D228Y突变强烈地增加了CEB1重排的频率。我们解释说,Pif1在滞后链DNA中是必不可少的,因为RPA本身能够阻止在滞后链合成过程中形成稳定的富含g的二级结构。值得注意的是,在rfa1-D228Y突变体中,Pif1的过表达挽救了CEB1在滞后链上的不稳定性,这表明Pif1也可以在滞后链上起作用。我们发现rfa1-D228Y(裂变酵母中的rpa1-D223Y)的作用在裂糖酵母中是保守的。最后,我们报道了在出芽酵母中RNase H1以dna依赖的方式与RPA相互作用,然而在pif1Δ和rfa1-D228Y突变体中观察到的RNase H1过表达并不能挽救CEB1的不稳定性。总的来说,这些结果为RPA在防止DNA二级结构形成和协调旨在解决这些结构的因子的作用方面的一般作用提供了新的见解。
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来源期刊
Cell Stress
Cell Stress Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (miscellaneous)
CiteScore
13.50
自引率
0.00%
发文量
21
审稿时长
15 weeks
期刊介绍: Cell Stress is an open-access, peer-reviewed journal that is dedicated to publishing highly relevant research in the field of cellular pathology. The journal focuses on advancing our understanding of the molecular, mechanistic, phenotypic, and other critical aspects that underpin cellular dysfunction and disease. It specifically aims to foster cell biology research that is applicable to a range of significant human diseases, including neurodegenerative disorders, myopathies, mitochondriopathies, infectious diseases, cancer, and pathological aging. The scope of Cell Stress is broad, welcoming submissions that represent a spectrum of research from fundamental to translational and clinical studies. The journal is a valuable resource for scientists, educators, and policymakers worldwide, as well as for any individual with an interest in cellular pathology. It serves as a platform for the dissemination of research findings that are instrumental in the investigation, classification, diagnosis, and therapeutic management of major diseases. By being open-access, Cell Stress ensures that its content is freely available to a global audience, thereby promoting international scientific collaboration and accelerating the exchange of knowledge within the research community.
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