Single-Stranded Annealing Induced by Re-Initiation of Replication Origins Provides a Novel and Efficient Mechanism for Generating Copy Number Expansion via Non-Allelic Homologous Recombination.
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE41259 | GEO | 2013/01/04
SECONDARY ACCESSION(S): PRJNA176386
REPOSITORIES: GEO
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Project description:Copy number expansions such as amplifications and duplications contribute to human phenotypic variation, promote molecular diversification during evolution, and drive the initiation and/or progression of various cancers. The mechanisms underlying these copy number changes are still incompletely understood, however. We recently demonstrated that transient, limited re-replication from a single origin in Saccharomyces cerevisiae efficiently induces segmental amplification of the re-replicated region. Structural analyses of such re-replication induced gene amplifications (RRIGA) suggested that RRIGA could provide a new mechanism for generating copy number variation by non-allelic homologous recombination (NAHR). Here we elucidate this new mechanism and provide insight into why it is so efficient. We establish that sequence homology is both necessary and sufficient for repetitive elements to participate in RRIGA and show that their recombination occurs by a single-strand annealing (SSA) mechanism. We also find that re-replication forks are prone to breakage, accounting for the widespread DNA damage associated with deregulation of replication proteins. These breaks appear to stimulate NAHR between re-replicated repeat sequences flanking a re-initiating replication origin. Our results support a RRIGA model where the expansion of a re-replication bubble beyond flanking homologous sequences followed by breakage at both forks in trans provides an ideal structural context for SSA–mediated NAHR to form a head-to-tail duplication. Given the remarkable efficiency of RRIGA, we suggest it may be an unappreciated contributor to copy number expansions in both disease and evolution. The arrays in this series are primary comparative genomic hybridizations to determine genomic changes after re-replication. Genomic DNA was purified from reference Saccharomyces cerevisiae cells or survivors of re-replication, differentially labeled with Cy3 and Cy5, and competitively hybridized to a spotted microarray containing ORF and intergenic PCR products. Cy5/Cy3 ratios (or the inverse in the case of dye swapped samples) are normalized so that the average ratio of all elements was 1 (except for arrays measuring re-replication induction where this ratio was set to 2). A small number of the arrays were used to determine the extent and location of re-replication under different conditions. For those, genomic DNA was purified from non-replicating and re-replicating cells and treated as above. Series contains a total of 333 hybridizations. Note that the VALUE field reported for all of the sample tables in this series are the log2 of the normalized Cy5/Cy3 (or Cy3/Cy5) ratio. To convert these into usable copy number profiles raise 2 to the indicated VALUE.
2013-01-04 | E-GEOD-41259 | 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.
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: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:Eukaryotic cells use numerous mechanisms to ensure that no segment of their DNA is re-replicated within a single cell cycle. Despite longstanding speculation that such tight regulation is needed to protect cells from genomic alterations, this notion has never been experimentally tested. Here we show that even just transient and limited re-replication in Saccharomyces cerevisiae can strongly induce the critical first step of gene amplification, increasing gene copy number from one to two or more. The amplified units, or amplicons, consist of large internal chromosomal segments that are bounded by Ty repetitive elements and are intrachromosomally arrayed at their endogenous locus in direct head-to-tail orientation. The presence of hybrid Ty elements at inter-amplicon junctions together with the dependence of amplification on RAD52 indicate that in budding yeast these re-replication-induced gene amplifications (RRIGA) are mediated by homologous recombination between re-replicated non-allelic repetitive elements. These results finally establish the importance of stringent replication control for genome stability and suggest that re-replication should now be considered as a possible contributor to gene copy number changes in fields as diverse as cancer biology, evolution, and human genetics.
2011-05-17 | GSE22018 | GEO
Project description:Eukaryotic cells use numerous mechanisms to ensure that no segment of their DNA is re-replicated within a single cell cycle. Despite longstanding speculation that such tight regulation is needed to protect cells from genomic alterations, this notion has never been experimentally tested. Here we show that even just transient and limited re-replication in Saccharomyces cerevisiae can strongly induce the critical first step of gene amplification, increasing gene copy number from one to two or more. The amplified units, or amplicons, consist of large internal chromosomal segments that are bounded by Ty repetitive elements and are intrachromosomally arrayed at their endogenous locus in direct head-to-tail orientation. The presence of hybrid Ty elements at inter-amplicon junctions together with the dependence of amplification on RAD52 indicate that in budding yeast these re-replication-induced gene amplifications (RRIGA) are mediated by homologous recombination between re-replicated non-allelic repetitive elements. These results finally establish the importance of stringent replication control for genome stability and suggest that re-replication should now be considered as a possible contributor to gene copy number changes in fields as diverse as cancer biology, evolution, and human genetics. The arrays in this series are primary comparative genomic hybridizations to determine genomic changes after re-replication or other genomic stresses. Genomic DNA was purified from reference Saccharomyces cerevisiae cells or survivors of re-replication or other genomic stresses, differentially labeled with Cy3 and Cy5, and competitively hybridized to a spotted microarray containing ORF and intergenic PCR products. Cy5/Cy3 ratios are normalized so that the average ratio of all elements was 1. A small number of the arrays were used to determine the extent and location of re-replication under different conditions. For those, genomic DNA was purified from non-replicating and re-replicating cells and treated as above. Series contains a total of 203 hybridizations.
2011-05-17 | E-GEOD-22018 | biostudies-arrayexpress
Project description:The sources of genome instability, a hallmark of cancer, remain incompletely understood. One potential source is DNA re-replication, which arises when the mechanisms that prevent re-initiation of replication origins within a single cell cycle are compromised. Using the budding yeast Saccharomyces cerevisiae, we previously showed that DNA re-replication is extremely potent at inducing gross chromosomal alterations and that this arises in part because of the susceptibility of re-replication forks to break. Here, we examine the ability of DNA re-replication to induce nucleotide level mutations. During normal replication these mutations are restricted by three overlapping error avoidance mechanisms: the nucleotide selectivity of replicative polymerases, their proofreading activity, and mismatch repair. Using lys2InsEA14, a frameshift reporter that is poorly proofread, we show that re-replication induces up to a 30x higher rate of frameshift mutations and that this mutagenesis is due to passage of the re-replication fork, not secondary to re-replication fork breakage. Re-replication can also induce comparable rates of frameshift and base substitution mutations in a more general mutagenesis reporter CAN1, when the proofreading activity of DNA polymerase ε is inactivated. Finally, we show that the induction of lys2InsEA14 frameshift mutations by re-replication is dependent on mismatch repair. These results suggest that the mismatch repair associated with re-replication is attenuated, although at most sequences DNA polymerase proofreading provides enough error correction to mitigate the mutagenic consequences. Thus, re-replication can facilitate nucleotide level mutagenesis in addition to inducing gross chromosomal alterations, broadening its potential role in genome instability.
2018-12-27 | GSE124382 | GEO
Project description:Heterochromatin contains repressively modified histones and replicates late in S phase of the cell cycle. Besides the shortage in replication origins, little is known about replication timing regulation in silenced regions. In Drosophila polytene cells, late replication results in under-replication and decreased DNA copy number in heterochromatic regions of the genome. The Suppressor of Under-replication (SUUR) protein controls this feature â?? in its absence the DNA polytenization level in most silenced regions is restored, however the repressive histone marks are lost. We hypothesized that SUUR regulates the re-establishment of repressive histone pattern during replication which results in delayed replication completion of heterochromatin. Measuring DNA copy number in mutants with disrupted repressive pathways, we found that under-replication is directly linked to repressive histone marks supply. DamID-seq and ChIP-seq experiments revealed that SuUR mutation does not affect the establishment of heterochromatin domains. Here, we identified a novel SUUR protein interaction (CG12018) that supports SUUR association with replication complex. SUUR loads onto replication forks shortly after the origin firing and participates in chromatin maintenance rather than its establishment. Thus, our findings provide comprehensive evidence that late replication in Drosophila is caused by the time-consuming process of replication-coupled repressive chromatin renewal. Examination of three chromatin proteins in slivary glands in presence or absence of SuUR mutation using DamID technique.
2016-01-10 | E-GEOD-74907 | biostudies-arrayexpress
Project description:Heterochromatin contains repressively modified histones and replicates late in S phase of the cell cycle. Besides the shortage in replication origins, little is known about replication timing regulation in silenced regions. In Drosophila polytene cells, late replication results in under-replication and decreased DNA copy number in heterochromatic regions of the genome. The Suppressor of Under-replication (SUUR) protein controls this feature â in its absence the DNA polytenization level in most silenced regions is restored, however the repressive histone marks are lost. We hypothesized that SUUR regulates the re-establishment of repressive histone pattern during replication which results in delayed replication completion of heterochromatin. Measuring DNA copy number in mutants with disrupted repressive pathways, we found that under-replication is directly linked to repressive histone marks supply. DamID-seq and ChIP-seq experiments revealed that SuUR mutation does not affect the establishment of heterochromatin domains. Here, we identified a novel SUUR protein interaction (CG12018) that supports SUUR association with replication complex. SUUR loads onto replication forks shortly after the origin firing and participates in chromatin maintenance rather than its establishment. Thus, our findings provide comprehensive evidence that late replication in Drosophila is caused by the time-consuming process of replication-coupled repressive chromatin renewal. Examination of H3K27me3 histone modification in 3 cell types in presence or absence of SuUR mutation.
2016-01-10 | E-GEOD-74908 | biostudies-arrayexpress