Topoisomerase I‐mediated cleavage at unrepaired ribonucleotides generates DNA double‐strand breaks. (1st February 2017)

• Record Type: Journal Article
• Authors: Huang, Shar‐yin N; Williams, Jessica S; Arana, Mercedes E; Kunkel, Thomas A; Pommier, Yves
• Is Part Of: EMBO journal. Volume 36:Number 3(2017)
• Page Start: 361
• Page End: 373
• Publication Date: 2017-02-01
• Subjects: double‐strand breaks -- homologous recombination -- ribonucleotide excision repair -- RNase H2 -- topoisomerase I; Molecular biology -- Periodicals; 572.805
• DOI: 10.15252/embj.201592426
• Languages: English
• ISSNs: 0261-4189

Abstract

Abstract: 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 Top1‐induced 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. Synopsis: Topoisomerase I (Top1) is known to generate single‐strand breaks at sites of newly incorporated and unrepaired ribonucleotides in DNA. Sequential Top1 cleavage on the opposite strand can directly lead to DNA double‐strand breaks (DSBs), causing replication stress and genome instability and requiring repair by homologous recombination. Top1 induces DSBs in yeast mutants lacking ribonucleotide excision repair by RNase H2. Top1‐induced DNA damage at ribonucleotide incorporation sites requires Rad51 and Rad52 for repair. In vitro, Top1 displays ribonuclease activity at the misincorporation site followed by Top1 cleavage events on the opposite DNA strand. Sequential Top1 cleavage generates DSBs whose ends are covalently linked to Top1, making them prone to recombination. Abstract : At sites of newly incorporated ribonucleotides, Top1 may not only induce single‐strand breaks but also sequentially cleave both DNA strands, causing genome damage that requires repair by homologous recombination.