qDSB-Seq: quantitative DNA double-strand break sequencing

Yingjie Zhu, Anna Biernacka, Benjamin Pardo, Norbert Dojer, Romain Forey, Magdalena Skrzypczak, Bernard Fongang, Jules Nde, Raziyeh Yousefi, Philippe Pasero, Krzysztof Ginalski, Maga Rowicka

Research output: Contribution to journalArticlepeer-review

Abstract

Sequencing-based methods for mapping DNA double-strand breaks (DSBs) allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. We propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induced spike-in DSBs by a site-specific endonuclease and used them to quantify labeled DSBs (e.g. using i-BLESS). Utilizing qDSB-Seq, we determined numbers of DSBs induced by a radiomimetic drug and various forms of replication stress, and revealed several orders of magnitude differences in DSB frequencies. We also measured for the first time Top1-dependent absolute DSB frequencies at replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.

Original languageEnglish (US)
JournalUnknown Journal
DOIs
StatePublished - Aug 11 2017

Keywords

  • Camptothecin
  • DNA double-stranded break
  • Fork barrier
  • Hydroxyurea
  • Quantitative DSB sequencing
  • Replication fork
  • Restriction enzyme
  • Zeocin

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)

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