Topoisomerase I-mediated cleavage at ribonucleotides generates DNA double-strand breaks
Topoisomerase I-mediated cleavage at ribonucleotides generates DNA double-strand breaks
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DOI：
10.15252/embj.201592426
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发表时间：
2017-02-01
期刊：
影响因子：
11.4
通讯作者：
Pommier, Yves
中科院分区：
文献类型：
--
作者：
Huang, Shar-yin N.;Williams, Jessica S.;Pommier, Yves
拓扑异构酶I （Top1）的核糖核酸酶活性导致核糖核苷酸位点上含有2‘,3’环磷酸的DNA刻痕。在这里，我们提供了遗传和生化证据，证明DNA双链断裂（DSBs）可以由基因组核糖核苷酸位点的Top1直接产生。我们发现，RNase H2缺失的酵母细胞Rad52灶频率升高，RNase H2和Rad52失活导致合成致死，RNase H2和RAD51的联合缺失导致生长缓慢和复制应激。重要的是，这些表型在TOP1的额外缺失后被修复，这意味着同源重组修复了TOP1在核糖核苷位点引起的损伤。我们从生物化学的角度证明，不可逆的dsb是由随后的Top1切割产生的，而不是由Top1诱导的核糖核苷酸位点上的DNA缺口。下拉实验中对Top1链DNA的分析显示，在RNase h2缺失的酵母细胞中，Top1与DNA末端共价连接，支持该模型。综上所述，当复制聚合酶结合的核糖核苷酸没有被RNase H2去除时，这些结果将Top1定义为dsb和基因组不稳定的来源。
Ribonuclease activity of topoisomerase I (Top1) causes DNA nicks bearing 2',3'-cyclic phosphates at ribonucleotide sites. Here, we provide genetic and biochemical evidence that DNA double-strand breaks (DSBs) can be directly generated by Top1 at sites of genomic ribonucleotides. We show that RNase H2-deficient yeast cells displayed elevated frequency of Rad52 foci, inactivation of RNase H2 and RAD52 led to synthetic lethality, and combined loss of RNase H2 and RAD51 induced slow growth and replication stress. Importantly, these phenotypes were rescued upon additional deletion of TOP1, implicating homologous recombination for the repair of Top1-induced damage at ribonuclelotide sites. We demonstrate biochemically that irreversible DSBs are generated by subsequent Top1 cleavage on the opposite strand from the Top1induced DNA nicks at ribonucleotide sites. Analysis of Top1-linked DNA from pull-down experiments revealed that Top1 is covalently linked to the end of DNA in RNase H2-deficient yeast cells, supporting this model. Taken together, these results define Top1 as a source of DSBs and genome instability when ribonucleotides incorporated by the replicative polymerases are not removed by RNase H2.