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Break-Seq to map DNA double stranded breaks in WT S. cerevisiae WT post treatment with six different replication inhibitors

ABSTRACT: Replication–transcription conflicts have long been identified as a source of genome instability, leading to DNA double-strand break (DSBs) formation at the site of encounter. The encounter between replication and transcription is significantly increased when cells encounter stress, such as by treatment with agents that alter either of these processes. Alterations in either of these processes increase the probability of their encounter, ultimately inducing DSBs. Previous studies with hydroxyurea (HU)-treated synchronous cell cultures showed that HU-induced DSBs are a result of replication-transcription collisions. Here, we sought to determine whether replication-transcription collision is a general phenomenon used by replication inhibitors to induce DSBs. In our study, we used six different replication inhibitors, HU, methyl methane sulfonate (MMS), camptothecin (CPT), actinomycin D (AD), doxorubicin (DOX), and methotrexate (MTX), each of which acts differently. The replication inhibitor-specific DSBs tend to be enriched at gene exons and are also well correlated with the origins of replication. Gene expression around these DSBs tends to be upregulated, thus increasing the probability of encounter between the two. These results support our hypothesis that DSBs result from replication-transcription conflicts. We also observed enrichment in the transcription-dependent histone mark deposition-H3K4me3 and H3K36me3 at one DSB locus. Taken together, these results suggest that replication inhibitor treatment induces the expression of certain genes, depositing active histone marks, which upon encountering the incoming replication fork result in collision. Overall, our study maps the genome-wide location of DSBs as well as the gene expression profile induced by different replication inhibitors and provides insight into the role of chromatin architecture in regulating genome integrity.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE234408 | GEO | 2025/12/31

REPOSITORIES: GEO

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Project description:We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation. Here we sought to determine whether all replication forks containing ssDNA 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 NextGen 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 greater distance in MEC1 cells than in mec1 cells during the 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 the said 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. Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage? We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs. We queried the yeast genome for gene expression after cells were treated with 200 mM hydroxyurea during S phase. Samples were collected from 1) cells synchronized in G1 phase by alpha factor; 2) cells released from G1 into medium containing 200 mM hydroxyurea for 1 h; 3) cells recovering in fresh medium without hydroxyurea for 30 and 60 min after the 1 h exposure to HU. These samples are referred to as G1, HU 1h, R30, and R60, respectively. The strains from which the samples were collected are indicated before the time point, e.g. mec1_G1 or MEC1_R30. Stranded mRNA libraries were prepared according to manufacturer's suggestion and sequenced on Illumina MiSeq with paired-end reads of 75 bp.

Project description:We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation. Here we sought to determine whether all replication forks containing ssDNA 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 NextGen 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 greater distance in MEC1 cells than in mec1 cells during the 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 the said 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. Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage? We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs. We queried the yeast genome for DSBs after cells were treated with 200 mM hydroxyurea during S phase. Samples were collected from 1) cells synchronized in G1 phase by alpha factor; 2) cells released from G1 into medium containing 200 mM hydroxyurea for 1 h; 3) cells recovering in fresh medium without hydroxyurea for 1 h after the 1 h exposure to HU. These samples are referred to as G1, HU 1h, and R 1h, respectively. The strains from which the samples were collected are indicated following the time point, e.g. G1_MEC1 or R 1h_mec1. The experiment with mec1 was done twice (Experiments A and B) and that with MEC1 was done once (Experiment C). In addition, a control experiment of in vitro digestion with BamHI using the G1_mec1 sample (G1_BamHI) was performed.

Project description:Many genotoxic agents that block replication fork progression to cause DNA replication stress also block transcription, which results in transcription stress. The 7SK-small nuclear ribonucleoprotein (7SK-snRNP) regulates and promotes RNA polymerase II activity in response to transcription blocks. Individual 7SK-snRNP components HEXIM1, LARP7 and MEPCE are also reported to either facilitate or inhibit processes relevant to replication stress: conflicts between transcription and replication and homologous recombination (HR) at double-strand breaks (DSBs). Here, we investigate potential roles of 7SK-snRNP components in the response to replication stress-inducing agents such as camptothecin and hydroxyurea. We report that HEXIM1 and LARP7 promote replication fork slowing in response to agents that cause both replication- and transcription stress, in a manner consistent with their canonical 7SK-snRNP functions. Our data suggest that this role in fork slowing is mainly through facilitating RAD51-mediated replication fork reversal rather than transcription-replication conflicts. HEXIM1 and LARP7 promote RAD51 recruitment to replication-associated DSBs or post-replicative gaps under conditions of transcription stress such as induced by camptothecin or BET inhibitors, while to a lesser extent supporting HR at direct DSBs and not at all in absence of transcription stress. Our data support that LARP7 roles during replication stress are independent of its reported interaction with BRCA1 and both LARP7 and HEXIM1 promote survival of replication stress-inducing agents, especially camptothecin. 7SK-snRNP components are not recruited to stressed replication forks and RNA polymerase II inhibition phenocopies loss of these proteins. Taken together, our data support a model where 7SK-snRNP modulation of RNA polymerase II activity helps facilitate RAD51 function under transcription stress conditions, thereby linking the cell responses to transcription- and replication stress.

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Break-Seq to map DNA double stranded breaks in WT S. cerevisiae WT post treatment with six different replication inhibitors

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