Loss of DNA replication control is a potent initiator of gene amplification
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE22018 | GEO | 2011/05/17
SECONDARY ACCESSION(S): PRJNA127435
REPOSITORIES: GEO
Similar Datasets
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: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.
2013-01-04 | GSE41259 | GEO
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:Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1/Timeless, a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Î mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of the FPC in regulation of telomere stability in cancer cells. Genome-wide distributions of Rad52 in wild type and in swi1 deletion in fission yeast The'SP1173_WT ChIP-seq' is an input sample (non-tagged data).
2016-02-25 | E-GEOD-74487 | biostudies-arrayexpress
Project description:Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, ChIP on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double deletion of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD51 single deletion. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed. Keywords: ChIP-chip ⢠The goal of the experiment Genome-wide localization of Rad52 binding sites in Saccharomyces cerevisiae ⢠Experimental factor Distribution of Rad52 on chromosome III, IV, and V and the right arm of chromosome VI Strain: wild type, rad53 mutant, and rad53 siz2 mutant (W303 background, expressing myc tagged protein from its native promoter) Cell condition 1: G1 arrest with alpha-factor Cell condition 2: HU treatment ⢠Experimental design ChIP analyses: ChIP using anti-c-Myc antibody. ChIP-chip analyses: In all cases, hybridization data of ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F) were used. ⢠Quality control steps taken Confirmation of several loci by quantitative real time PCR. Wild type cells expressing non-tagged Rad52 were used as a negative control of DNA amplification.
2010-05-18 | E-GEOD-14761 | biostudies-arrayexpress
Project description:To achieve faithful replication of the genome once in each cell cycle, re-initiation of S-phase is prevented in G2 and origins are restricted from re-firing within S-phase. We have investigated the block to re-replication during G2 in fission yeast. The DNA synthesis that occurs when G2/M cyclin dependent kinase (CDK) activity is depleted has been assumed to be repeated rounds of S-phase without mitosis but this has not been demonstrated to be the case. In a normal mitotic S-phase, the genome is uniformly replicated with no areas amplified relative to other regions, so there is an equal copy number of each portion of the genome. To determine whether equal rounds of replication or local amplification occurred in the cdc13 s/o strain we assayed genomic DNA from cells which had increased their DNA content to 16C-32C. Using microarrays, we measured the relative DNA content across the genome, using DNA from control G1-arrested cells as a reference. This experiment revealed that replication was essentially equal across the genome, with no region becoming significantly amplified to a higher copy number than any other. Since the genome was found to be essentially evenly replicated as would be expected if each round of replication corresponded to a normal S-phase, we investigated whether this was the result of a normal replication program at the level of origin firing. We asked whether S-phase origins are used to reduplicate the genome, and whether origins are used with the same efficiency as in wild type cells. We identified the origins utilized in the first endoreduplication cycle of cdc13 s/o using microarray analyses of cells treated with 11 mM HU. Samples were taken at 5 hours when most cells of the culture not treated with HU had undergone a doubling in DNA content. A total of 799 origins were identified, a number roughly similar to the 904 identified in a normal S-phase. We show here that on G2/M CDK depletion in G2, repeated S-phases are induced. Mostly normal mitotic S-phase origins were utilized although at different efficiencies, and replication was essentially equal across the genome. We conclude that CDK inhibits re-initiation of S-phase during G2, and if G2/M CDK is depleted replication results from induction of a largely normal S-phase program with only small differences in origin usage and efficiency.
2009-05-13 | E-MTAB-105 | biostudies-arrayexpress
Project description:Multiple pathways prevent DNA replication from occurring more than once per cell cycle. These pathways block re-replication by strictly controlling the activity of pre-replication complexes, which assemble at specific sites in the genome called origins. Here we show that mutations in the homologous histone 3 lysine 27 (H3K27) monomethyltransferases, ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6, lead to re-replication of specific genomic locations. The vast majority of these locations correspond to transposons and other repetitive and silent elements of the Arabidopsis genome. These sites also correspond to high levels of H3K27 monomethylation, and mutation of the catalytic SET domain is sufficient to cause the re-replication defect. Mutation of ATXR5 and ATXR6 also causes upregulation of transposon expression and has pleiotropic effects on plant development. These results uncover a novel pathway that prevents over-replication of heterochromatin in Arabidopsis. Examination of genomic DNA content in endoreduplicating nuclei in wild type and atxr atxr6 mutant plants
2010-07-21 | E-GEOD-22411 | biostudies-arrayexpress
Project description:Identification of sites of replication initiation by copy number analysis, replicated DNA vs unreplicated DNA
2018-05-31 | GSE98446 | GEO
Project description:At the organismal level, genome rearrangements are usually deleterious and are often associated with disease. Yet, on an evolutionary scale, they can be beneficial as they provide for rapid genetic diversification. DNA lesions, particularly double-strand breaks (DSBs), are sources of genome instability that can be rectified by various repair processes. Homologous recombination (HR) is highly effective at protecting the genome from DSBs and provides for accurate repair between sister chromatids and homologous chromosomes. Here we show that although random DSBs induced by ionizing radiation in yeast chromosomes are repaired efficiently by HR in G-2 diploid cells, rearrangements are frequent. The chromosome aberrations (ABs) primarily resulted from recombination between Ty retrotransposable elements, the most abundant class of dispersed repetitive DNAs in the genome, while some rearrangements involved other classes of repetitive DNA. Few, if any, of the ABs could be attributed to nonhomologous end-joining (NHEJ). We conclude that only those few DSBs that fall at or near the 3-5% of the genome composed of repetitive DNA elements are effective at generating rearrangements, while other lesions that appear in unique (single copy) sequences are correctly repaired. Thus, by successfully competing with repair that normally occurs between large homologous chromosomal DNAs, the combination of repetitive elements and DSBs provides genome plasticity and a rich source of evolutionary opportunities. Keywords: CGH-array
2007-02-08 | GSE6984 | GEO
Project description:At the organismal level, genome rearrangements are usually deleterious and are often associated with disease. Yet, on an evolutionary scale, they can be beneficial as they provide for rapid genetic diversification. DNA lesions, particularly double-strand breaks (DSBs), are sources of genome instability that can be rectified by various repair processes. Homologous recombination (HR) is highly effective at protecting the genome from DSBs and provides for accurate repair between sister chromatids and homologous chromosomes. Here we show that although random DSBs induced by ionizing radiation in yeast chromosomes are repaired efficiently by HR in G-2 diploid cells, rearrangements are frequent. The chromosome aberrations (ABs) primarily resulted from recombination between Ty retrotransposable elements, the most abundant class of dispersed repetitive DNAs in the genome, while some rearrangements involved other classes of repetitive DNA. Few, if any, of the ABs could be attributed to nonhomologous end-joining (NHEJ). We conclude that only those few DSBs that fall at or near the 3-5% of the genome composed of repetitive DNA elements are effective at generating rearrangements, while other lesions that appear in unique (single copy) sequences are correctly repaired. Thus, by successfully competing with repair that normally occurs between large homologous chromosomal DNAs, the combination of repetitive elements and DSBs provides genome plasticity and a rich source of evolutionary opportunities. Keywords: Band-array
2007-02-10 | GSE6991 | GEO