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Topoisomerase I‐mediated cleavage at unrepaired ribonucleotides generates DNA double‐strand breaks
Author(s) -
Huang Sharyin N,
Williams Jessica S,
Arana Mercedes E,
Kunkel Thomas A,
Pommier Yves
Publication year - 2017
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.15252/embj.201592426
Subject(s) - biology , rad52 , rnase p , ribonucleotide , rnase mrp , rnase h , dna , dna repair , microbiology and biotechnology , genome instability , dna polymerase , rad51 , homologous recombination , replication protein a , dna replication , dna damage , biochemistry , rna , dna binding protein , nucleotide , gene , transcription factor
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.