DYNAMICS OF REPLICATION ORIGIN OVER-ACTIVATION
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE172417 | GEO | 2021/06/01
REPOSITORIES: GEO
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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.
2014-03-15 | E-GEOD-55420 | biostudies-arrayexpress
Project description:Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation 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, origins re-initiate with diverse efficiencies when these mechanisms are disabled, and this diversity cannot be explained by differences in the efficiency or timing of origin initiation during normal S phase replication. This observation raises the possibility of an additional layer of replication control that can differentially regulate re-initiation at distinct origins. 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 ~60 bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 reassociates 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. Our findings identify 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 compromised. 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.
2014-03-15 | GSE55420 | GEO
Project description:Cell cycle progression in mammals is modulated by two ubiquitin ligase complexes, CRL4 and SCF, which facilitate degradation of chromatin substrates involved in the regulation of DNA replication. One member of the CRL4 complex, RepID (DCAF14/PHIP), is a WD40-containing protein that selectively binds and activates a group of replication origins. We found that RepID recruits the CRL4 complex to chromatin prior to DNA synthesis, thus playing a crucial architectural role in the proper licensing of chromosomes for replication. In the absence of RepID, cells rely on the alternative ubiquitin ligase, SKP2-containing SCF, to progress through the cell cycle. RepID depletion markedly increases cellular sensitivity to SKP2 inhibitors, which triggered massive genome re-replication. Both RepID and SKP2 interact with distinct, non-overlapping groups of replication origins, suggesting that selective interactions of replication origins with specific CRL components execute the DNA replication program and maintain genomic stability by preventing re-initiation of DNA replication.
2018-06-27 | GSE114703 | GEO
Project description:During S-phase of the eukaryotic cell cycle, controls exist to ensure that replication origins fire only once and become inactivated after traverse of a replication fork. Failure of these controls results in local amplification of the genome which can lead to gene copy increases important for both evolutionary change and for somatic genetic diseases such as cancer. It is not known what characteristics of replication origins might predispose them to escape from these controls. We have investigated this problem in the fission yeast Schizosaccharomyces pombe by characterizing regions of the genome which become amplified when the DNA synthesis initiation factors cdc18 (cdc6) and cdt1 are co-overexpressed and by defining origins of replication responsible for local amplification. We find that a single origin can be necessary but not sufficient to induce ectopic local amplification and that origins likely to escape the regulation of one firing per cell cycle are among the most AT-rich, efficient and early firing in the genome, and are embedded in long intergenes. Replication origins with these features may be more prone to re-fire within a round of replication potentiating genome instability.
2010-08-13 | E-MTAB-139 | biostudies-arrayexpress
Project description:The budding yeast telomere binding protein Rif1 (Rap1-interacting factor 1) plays an evolutionarily conserved role in the control of DNA replication timing, which operates through an interaction with the PP1 phosphatase. Rif1-PP1 has been proposed to inhibit origin firing by reversing the phosphorylation of key targets involved in replication initiation. However, it is not yet known if Rif1 binds directly to the replication origins that it controls. Here we show that in unperturbed yeast cells Rif1 primarily regulates late-replicating telomere-proximal origins. Using Chromatin Endogenous Cleavage (ChEC)-seq, we find that Rif1 is robustly detected at many late-replicating origins that we identify as targets of its inhibitory action. Abrogation of Rif1 telomere binding, through mutation of its Rap1 binding module, leads to increased Rif1 binding and late origin inhibition elsewhere in the genome. Our results support a model whereby Rif1 inhibits replication initiation by binding directly at origins, most of which are near telomeres, where Rif1 is concentrated through its interaction with telomere-bound Rap1 protein.
2018-04-24 | GSE97953 | GEO
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:Mammalian DNA replication starts at distinct chromosomal sites in a tissue-specific pattern coordinated with transcription, but previous studies have not yet identified a chromatin modification that correlates with the initiation of DNA replication. This submission is associated with a paper in which we report that replication initiation events are associated with a high frequency of methylation of histone H3 on lysine K79 (H3K79Me2 and H3K79Me3). H3K79Me2-containing chromatin exhibited the highest enrichment of replication initiation events observed in a single chromatin modification. Importantly, H3K79 methylation was enriched in chromatin containing a replicator (a DNA sequence capable of initiating DNA replication), but not in chromatin containing a mutant replicator that could not initiate replication. The association of H3K79Me2 with replication initiation sites was independent and not synergistic with other chromatin modifications. H3K79 methylation exhibited a wider distribution and greater abundance during S-phase, but regions of chromatin that were only modified during S-phase were not enriched in replication initiation events. In addition, the paper shows that depletion of DOT1L, the sole enzyme responsible for H3K79 methylation, triggered limited genomic over-replication. These data are consistent with the hypothesis that methylation of H3K79 associates with replication origins and marks replicated chromatin during S-phase to prevent re-replication and preserve genomic stability. Evaluation of HeK79 methylation in chromatin samples from cell cycle fractionated K562 leukemia cells. Unsyncrhonized untreated cultures of K562 cells were fractionated by size using centrifugal elutriation. Chromatin was isolated and subject to ChIP-Seq with antibodies directed against dimethylated and trimethylated lysine on histone H3.
2013-06-06 | E-GEOD-35294 | biostudies-arrayexpress
Project description:DNA replication must be tightly controlled during each cell cycle to prevent unscheduled replication and ensure proper genome maintenance. The currently known controls that prevent re-replication act redundantly to inhibit pre-Replicative Complex (pre-RC) assembly outside of the G1 phase of the cell cycle. We have analyzed the effects of re-replication on the S. cerevisiae genome using a combination of Comparitive Genomic Hybridization (CGH) of re-replicating strains and Genome-Wide Location Analysis of pre-RC components. These data indicate which sites in the genome assemble pre-RCs under re-replication conditions, which sites undergo re-initiation and the extent of re-replication. Keywords: comparative genomic hybridization, ChIP-chip, re-replication, DNA replication, pre-RC
2006-03-20 | GSE4487 | GEO
Project description:Phosphorylated SIRT1 associates with replication origins to prevent excess replication initiation and preserve genomic stability
| PRJNA369608 | ENA
Project description:Mammalian chromosome replication starts from distinct sites, but the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. We identified a replication initiation determinant (RepID) protein that binds a subset of replication initiation sites. A large fraction of RepID binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from Rep-ID bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication initiation events. These observations are consistent with a model suggesting that RepID facilitates replication initiation at a distinct group of human replication origins. Nascent strands were purified with the lambda exonuclease methods from HCT116 cells and sequenced. Chromatin from unsyncrhonized untreated cultures of U2OS cells was subjected to ChIP-Seq with antibody directed against RepID/PHIP
2016-06-02 | E-GEOD-80298 | biostudies-arrayexpress