Unknown,Transcriptomics,Genomics,Proteomics

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Loss of DNA replication control is a potent initiator of gene amplification

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Brian Green

PROVIDER: E-GEOD-22018 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Loss of DNA replication control is a potent inducer of gene amplification.

Green Brian M BM Finn Kenneth J KJ Li Joachim J JJ

Science (New York, N.Y.) 20100801 5994

Eukaryotic cells use numerous mechanisms to ensure that no segment of their DNA is inappropriately re-replicated, but the importance of this stringent control on genome stability has not been tested. Here we show that re-replication in Saccharomyces cerevisiae can strongly induce the initial step of gene amplification, increasing gene copy number from one to two or more. The resulting amplicons consist of large internal chromosomal segments that are bounded by Ty repetitive elements and are intr ...[more]

PMID: 20724634

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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.

Project description:Background: Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation events can generate genomic alterations with extraordinary frequency. To minimize the probability of re-initiation from so many origins, cells use a battery of regulatory mechanisms that reduce the activity of replication initiation proteins. Given the global nature of these mechanisms, it has been presumed that all origins are inhibited identically. However, transiently disabling these mechanisms causes replication origins to re-initiate with diverse efficiencies, which do not correlate with known differences in the efficiency or timing of origin initiation during normal DNA replication. These observations suggest an additional local layer of replication control that can differentially influence how re-initiation is regulated at distinct origins. Principal Findings: We have identified novel genetic elements that are necessary for preferential re-initiation of two origins and sufficient to confer preferential re-initiation on heterologous origins when the control of re-initiation is partially deregulated. The elements do not enhance the S phase timing or efficiency of adjacent origins and thus are specifically acting as re-initiation promoters (RIPs). We have mapped the two RIPs to ~60bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 re-associates both with origins that preferentially re-initiate and origins that do not, suggesting that the RIP elements can overcome a block to re-initiation imposed after Mcm2-7 associates with origins. Conclusions/Significance: We have uncovered a local level of control in the block to re-initiation. This local control creates a complex genomic landscape of re-replication potential that is revealed when global mechanisms preventing re-replication are peeled away. Hence, if re-replication does contribute to genomic alterations, as has been speculated for cancer cells, some regions of the genome may be more susceptible to these alterations than others. 142 array CGH experiments are presented. Each experiment is done in experimental replicate. Cells grown as described in Richardson CD and Li JJ PLoS Genetics 2014. Ch1 samples taken from replicating (S phase) or re-replicating (M phase + 3/6 hour induced). Ch2 samples taken from M phase arrested DNA.

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Loss of DNA replication control is a potent initiator of gene amplification

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Loss of DNA replication control is a potent inducer of gene amplification.

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