Genomic DNA sequencing of budding yeast strains
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE114290 | GEO | 2018/07/16
REPOSITORIES: GEO
Similar Datasets
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.
2015-01-12 | E-GEOD-63516 | biostudies-arrayexpress
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.
2015-01-12 | E-GEOD-58808 | biostudies-arrayexpress
Project description:Faithful duplication of DNA is essential for the maintenance of genomic stability in all organisms. DNA synthesis proceeds bi-directionally with continuous synthesis of leading strand DNA and discontinuous synthesis of lagging strand DNA. Herein, we describe a method of enriching and Sequencing of Protein-Associated Nascent strand DNA (eSPAN) to detect whether a protein binds the leading- and lagging-strands of DNA replication forks. We show that Pol-epsilon, PCNA, Cdc45, Mcm6 and Mcm10 preferentially associate with leading strands, whereas Pol-alpha, Pol32, Pol-delta, Rfa1 and Rfc1 associate with lagging strands of hydroxyurea (HU)-stalled replication forks. In contrast, PCNA is enriched at lagging strands of normal replication forks in wild type cells and HU-stalled forks in cells lacking Elg1. These studies demonstrate a strategy to reveal proteins at leading and lagging strands of DNA replication forks, and suggest that the unloading of PCNA from lagging strands of HU-stalled replication forks helps maintain genome integrity.
2014-10-29 | GSE52614 | GEO
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.
2015-01-12 | GSE63516 | GEO
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.
2015-01-12 | GSE58808 | GEO
Project description:Faithful duplication of DNA is essential for the maintenance of genomic stability in all organisms. DNA synthesis proceeds bi-directionally with continuous synthesis of leading strand DNA and discontinuous synthesis of lagging strand DNA. Herein, we describe a method of enriching and Sequencing of Protein-Associated Nascent strand DNA (eSPAN) to detect whether a protein binds the leading- and lagging-strands of DNA replication forks. We show that Pol-epsilon, PCNA, Cdc45, Mcm6 and Mcm10 preferentially associate with leading strands, whereas Pol-alpha, Pol32, Pol-delta, Rfa1 and Rfc1 associate with lagging strands of hydroxyurea (HU)-stalled replication forks. In contrast, PCNA is enriched at lagging strands of normal replication forks in wild type cells and HU-stalled forks in cells lacking Elg1. These studies demonstrate a strategy to reveal proteins at leading and lagging strands of DNA replication forks, and suggest that the unloading of PCNA from lagging strands of HU-stalled replication forks helps maintain genome integrity. We synchronized yeast cells at G1 and released into early S phase in the presence of BrdU, a nucleotide analog that can be incorporated into newly synthesized DNA strand, and hydroxyurea (HU), a ribonucleotide reductase inhibitor. HU has no effect on initiation of DNA replication at early replication origins, but inhibit late replication firing. In addition, replication forks are stalled due to depletion of dNTPs. We then performed chromatin-immunoprecipitation of 12 proteins of interest following a standard procedure. Protein-bound DNAs were then reverse-crosslinked and double strand DNA was denatured. Nascent DNA was enriched by immunoprecipitation using anti-BrdU antibodies. The recovered ssDNA was first marked with ligation to one oligo at 3M-bM-^@M-^Y end before conversion to dsDNA for library preparation and sequencing. In this way, the directionality of ssDNA and therefore strand information of each sequenced DNA were known. The sequencing tag was mapped to both Watson (red) and Crick (blue) strands of the reference genome. In addition to ChIP-eSPAN, we also performed BrdU-IP and single strand DNA sequence (BrdU-ssSeq) and protein ChIP followed by single-strand DNA sequencing (ChIP-ssSeq) for each corresponding ChIP-eSPAN experiment. We also performed Mcm4 and Mcm6 ChIP-seq using cells synchronized at G1 phase of the cell cycle for identification of replication origins in comparison with published dataset. Some protein ChIP-ssSeq and ChIP-eSPAN experiments were repeated and the data fits well each other. Therefore, we did not repeat all protein ChIP-ssSeq and ChIP-eSPAN experiments.
2014-10-29 | E-GEOD-52614 | biostudies-arrayexpress
Project description:Analysis of the response to hydroxyurea in a yeast aft1 mutant strain compared to wild-type strain (BY4741 or BY4742 backgrounds). Cells were grown in YPD rich medium containong 200 mM hydroxyurea (HU) for 2 hours. Analysis of the response to hydroxyurea in a yeast aft1aft2 mutant strain (Y18aft2d) compared to wild-type strain (CM3260). Cells were grown in YPD rich medium containing 200 mM hydroxyurea (HU) for 2 hours. Gene expression changes due to the aft1 mutation were also analysed in absence of HU (BY4741 background). Analysis of mid-term response to hydroxyurea in a yeast wild-type strain (W303a). Cells were grown in YPD rich medium containing 200 mM hydroxyurea (HU) for 2 hours. Gene expression changes were analysed compared to the same strain grown in the same medium without HU. Keywords: repeat sample
2005-12-01 | GSE1914 | GEO
Project description:R-loops represent a major source of replication stress but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest and restart in S. cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate normally their chromosomes under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Indeed, these cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative step in resuming replication. Similar impairments in nascent DNA resection at HU-arrested forks were observed in human cells lacking RNase H2. However, the addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation, fully restored fork resection. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.
2023-10-14 | GSE215896 | GEO