Audoynaud Charlotte, Schirmeisen Kamila, Ait Saada Anissia, Gesnik Armelle, Fernández-Varela Paloma, Boucherit Virginie, Ropars Virginie, Chaudhuri Anusha, Fréon Karine, Charbonnier Jean-Baptiste, Lambert Sarah A E
Institut Curie, Université PSL, CNRS UMR3348, 91400 Orsay, France; Université Paris-Saclay, CNRS UMR3348, 91400 Orsay, France; Ligue Nationale Contre le cancer (équipe labélisée), Orsay, France.
Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France.
Mol Cell. 2023 Apr 6;83(7):1061-1074.e6. doi: 10.1016/j.molcel.2023.02.008. Epub 2023 Mar 2.
Nonhomologous end-joining (NHEJ) factors act in replication-fork protection, restart, and repair. Here, we identified a mechanism related to RNA:DNA hybrids to establish the NHEJ factor Ku-mediated barrier to nascent strand degradation in fission yeast. RNase H activities promote nascent strand degradation and replication restart, with a prominent role of RNase H2 in processing RNA:DNA hybrids to overcome the Ku barrier to nascent strand degradation. RNase H2 cooperates with the MRN-Ctp1 axis to sustain cell resistance to replication stress in a Ku-dependent manner. Mechanistically, the need of RNaseH2 in nascent strand degradation requires the primase activity that allows establishing the Ku barrier to Exo1, whereas impairing Okazaki fragment maturation reinforces the Ku barrier. Finally, replication stress induces Ku foci in a primase-dependent manner and favors Ku binding to RNA:DNA hybrids. We propose a function for the RNA:DNA hybrid originating from Okazaki fragments in controlling the Ku barrier specifying nuclease requirement to engage fork resection.
非同源末端连接（NHEJ）因子在复制叉保护、重启和修复过程中发挥作用。在此，我们确定了一种与RNA:DNA杂交体相关的机制，该机制在裂殖酵母中建立了NHEJ因子Ku介导的对新生链降解的屏障。核糖核酸酶H（RNase H）活性促进新生链降解和复制重启，其中RNase H2在处理RNA:DNA杂交体以克服Ku对新生链降解的屏障方面发挥着重要作用。RNase H2与MRN-Ctp1轴协同作用，以Ku依赖的方式维持细胞对复制应激的抗性。从机制上讲，在新生链降解过程中对RNaseH2的需求需要引发酶活性，该活性允许建立Ku对核酸外切酶1（Exo1）的屏障，而冈崎片段成熟的受损会增强Ku屏障。最后，复制应激以引发酶依赖的方式诱导Ku聚集，并促进Ku与RNA:DNA杂交体结合。我们提出，源自冈崎片段的RNA:DNA杂交体在控制Ku屏障方面具有功能，该屏障指定了参与叉状切除所需的核酸酶。
