title: Topoisomerase I-mediated cleavage at unrepaired ribonucleotides generates DNA double-strand breaks.
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.
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA.
authors.forename: Shar-Yin N
authors.initials: SN
authors.lastname: Huang
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, USA.
authors.forename: Jessica S
authors.initials: JS
authors.lastname: Williams
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, USA.
authors.forename: Mercedes E
authors.initials: ME
authors.lastname: Arana
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kunkel
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA pommier@nih.gov.
authors.forename: Yves
authors.identifier: 0000-0002-3108-0758
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D004247:DNA / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D017353:Gene Deletion; D051135:Rad51 Recombinase / Q000378:metabolism; D051721:Rad52 DNA Repair and Recombination Protein / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; C091139:ribonuclease HII; D016914:Ribonuclease H; D004264:DNA Topoisomerases, Type I
keywords: RNase H2; double‐strand breaks; homologous recombination; ribonucleotide excision repair; topoisomerase I