title: Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription.
abstract: We have previously demonstrated that in Saccharomyces cerevisiae replication, checkpoint inactivation via a mec1 mutation leads to chromosome breakage at replication forks initiated from virtually all origins after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Here we sought to determine whether all replication forks containing single-stranded DNA gaps have equal probability of producing double-strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-seq, that combines our previously described DSB labeling with next generation sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed a greater distance in MEC1 cells than in mec1 cells during recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of those transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. We further propose that replication inhibitors can induce unscheduled encounters between replication and transcription and give rise to distinct patterns of chromosome fragile sites.
authors.affiliation: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Hoffman
authors.affiliation: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: McCulley
authors.affiliation: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Haarer
authors.affiliation: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210, USA.
authors.forename: Remigiusz
authors.initials: R
authors.lastname: Arnak
authors.affiliation: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210, USA fengw@upstate.edu.
authors.forename: Wenyi
authors.initials: W
authors.lastname: Feng
mesh_terms: D002453:Cell Cycle / Q000235:genetics; D019457:Chromosome Breakage; D002873:Chromosome Fragility / Q000187:drug effects*; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D059014:High-Throughput Nucleotide Sequencing; D006918:Hydroxyurea / Q000494:pharmacology*; D007477:Ions / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics; D008670:Metals / Q000378:metabolism; D018741:Replication Origin; D013312:Stress, Physiological; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015003:Yeasts / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: D007477:Ions; D026901:Membrane Transport Proteins; D008670:Metals; D014157:Transcription Factors; D006918:Hydroxyurea