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Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions [mRNA-seq]

ABSTRACT: DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineered genetic systems in budding yeast to induce unscheduled replication in the G1-phase of the cell cycle. Unscheduled G1 replication initiated at canonical S-phase origins. We quantified the composition of replisomes in G1- and S-phase and identified firing factors, polymerase alpha, and histone supply as factors that limit replication outside S-phase. G1 replication per se did not trigger cellular checkpoints. Subsequent replication during S-phase, however, resulted in over-replication and led to chromosome breaks and chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA indicating head-to-tail replication collisions as a key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induced an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE208589 | GEO | 2022/11/08

REPOSITORIES: GEO

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Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions [ChIP-seq]

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AP-MS of replisomes from S. cerevisiae in S phase and after unscheduled G1 replication

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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

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SETD3-mediated histidine methylation of MCM7 regulates DNA replication by facilitating chromatin loading of MCM

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Effect of mini-chromosome maintenance protein 2 deficiency on the locations of DNA replication origins

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Replication origin mapping of Cdc13-Cdc2 cells in different G1 extension

Project description:Identification of sites of replication initiation by copy number analysis, comparing samples before and after entry into S phase. Experiments were performed in Cdc13-Cdc2 strain in which G1 phase has been delayed by 5 or 15 minutes.

2018-02-04 | GSE88712 | GEO

A new MCM modification cycle regulates DNA replication initiation

Project description:The hexameric DNA helicase MCM (Mcm2-7) is a central regulatory target in eukaryotic replication. Chromatin-bound MCM is kept inactive during G1 phase and subsequently activated in S phase to initiate replication. During this transition, the only known chemical change on the Mcm2-7 proteins is the gain of multi-site phosphorylation that promotes recruitment of co-factors. As replication initiation is tied intimately to multiple biological cues, additional changes on these proteins can provide a second regulatory point. Here we describe a new MCM modification cycle mediated by SUMO that enables a negative regulation of replication initiation. We show that all MCM subunits undergo sumoylation upon loading at origins in G1 phase prior to MCM phosphorylation. Then bulk MCM sumoylation is lost as MCM phosphorylation rises. The pattern of MCM sumoylation suggests a negative role in replication. Indeed, increasing MCM sumoylation delays genome-wide replication initiation. Mechanistically, this is partly due to enhancing the recruitment of a conserved phosphatase that delays MCM phosphorylation and activation. By revealing a new MCM modification cycle and its role in replication, our findings suggest a new model, in which MCM sumoylation counterbalances kinase-based regulation to ensure accurate control of replication initiation.

2016-02-01 | GSE70407 | GEO

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