S-phase checkpoint regulations that preserve replication and chromosome integrity upon dNTP depletion

Giannattasio, Michele; Branzei, Dana

doi:10.1007/s00018-017-2474-4

Cited by 60 publications

(77 citation statements)

References 184 publications

(266 reference statements)

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“…A number of different mechanisms have been put forward to explain how the multifaceted S-phase checkpoint contributes to genome protection 87 . Following depletion of dNTPs, this highly conserved checkpoint kinase pathway preserves the functionality and structure of stalled DNA replication forks and prevents chromosome fragmentation.…”

Section: Discussionmentioning

confidence: 99%

H3K4 methylation at active genes mitigates transcription-replication conflicts during replication stress

et al. 2020

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Transcription-replication conflicts (TRCs) occur when intensive transcriptional activity compromises replication fork stability, potentially leading to gene mutations. Transcriptiondeposited H3K4 methylation (H3K4me) is associated with regions that are susceptible to TRCs; however, the interplay between H3K4me and TRCs is unknown. Here we show that H3K4me aggravates TRC-induced replication failure in checkpoint-defective cells, and the presence of methylated H3K4 slows down ongoing replication. Both S-phase checkpoint activity and H3K4me are crucial for faithful DNA synthesis under replication stress, especially in highly transcribed regions where the presence of H3K4me is highest and TRCs most often occur. H3K4me mitigates TRCs by decelerating ongoing replication, analogous to how speed bumps slow down cars. These findings establish the concept that H3K4me defines the transcriptional status of a genomic region and defends the genome from TRC-mediated replication stress and instability.

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Section: Discussionmentioning

confidence: 99%

H3K4 methylation at active genes mitigates transcription-replication conflicts during replication stress

et al. 2020

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“…This would support a model in which stressed forks that have generated sufficient ssDNA for Rad53 activation in the wake of CMG may also inhibit normal fork progression in trans (Figure S11A). Such a mechanism may explain the global slowing of replication forks in yeast cells treated with HU and provide an additional strategy to help preserve dNTP levels during replication stress 28 . While Mec1-Ddc2-dependent Rad53 activation is bypassed here by the use of autophosphorylated recombinant Rad53 77 , reconstitution of this pathway will provide a gateway to differentiate between global and local Rad53 effects in the future.…”

Section: Discussionmentioning

confidence: 99%

“…Single-stranded DNA (ssDNA) generated in the wake of uncoupled CMG plays an important physiological role by providing a platform for the activation of the apical checkpoint kinase, Mec1-Ddc2/ATR-ATRIP (budding yeast/vertebrates) 26,27 . Downstream activation of the effector kinase, Rad53/CHK1, elicits a host of responses aimed at preserving genome integrity during replication stress, including cell cycle arrest, increased dNTP levels, nuclease inhibition, and inhibition of origin firing [28][29][30] .…”

Section: Introductionmentioning

confidence: 99%

Rad53 controls DNA unwinding after helicase-polymerase uncoupling at DNA replication forks

Devbhandari

Remus

2019

Preprint

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The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Using the reconstituted budding yeast DNA replication system, we demonstrate that Pol e variants harboring catalytic point mutations in the Pol2 polymerase domain, contrary to Pol2 polymerase domain deletions, inhibit DNA synthesis at replication forks by displacing Pol d from PCNA/primer-template junctions, causing excessive DNA unwinding by the replicative DNA helicase, CMG, uncoupled from DNA synthesis. Mutations that suppress the inhibition of Pol d by Pol e restore viability in Pol2 polymerase point mutant cells. We also observe uninterrupted DNA unwinding at replication forks upon dNTP depletion or chemical inhibition of DNA polymerases, demonstrating that leading strand synthesis is not tightly coupled to DNA unwinding by CMG. Importantly, the Rad53 kinase controls excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork-stabilization by the replication checkpoint. RESULTS Regulated replication of nucleosome-free plasmid templates in vitroPol2 polymerase point mutations, unlike Pol2 catalytic domain deletions, are lethal 8 . Yet, fork rates in the presence of a catalytic mutant of Pol e (Pol e D640A ) or Pol e lacking the catalytic domain (Pol e Dcat ) appeared to be similar in vitro 13,37 . Therefore, to investigate potential differences between these Pol e variants, we decided to characterize them side-by-side using the reconstituted origin-dependent budding yeast DNA replication system 13,37 . We had previously demonstrated that nucleosomes limit Okazaki fragment length in vitro by restricting strand-displacement synthesis by Pol d 37 . However, using higher salt and lower Pol d concentrations we observe Okazaki fragments with a physiological length distribution also on naked DNA templates ( Figure S1).The modified conditions support efficient maturation of nascent strands by Cdc9 and Fen1 and normal replication of plasmid DNA templates (Figures 1A+B). In the presence of Top2, the two predominant ligation products were full-length or close to full-length linears (ssL), and covalently closed circles (ccc) (Figures 1A).The latter are an expected product of termination, while the former may result from fork stalling upon convergence 38 . Linear full-length or close to full-length daughter strands were also generated in the presence of Top1. As expected, due to the inability of Top1 to decatenate double-stranded DNA molecules, circular daughter strands remain topologically linked (cat). The ratio of circular to linear daughter strands was reduced in the presence of Top1 compared to Top2, indicating that Top2 has a greater proficiency in promoting replication termination. On average, we observe a termination efficiency of ~30 % in the presence of Top1 and ~45 % in the presence of Top2 within 45 minutes after origin firing on 4.8 kbp plasmid templates (Figure S2A).In the absence of topoisomerase, replication forks stalled ~ ...

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“…Replication stress, for example, caused by DNA lesions, conflicts between DNA and RNA polymerase, or low levels of deoxynucleotide triphosphates (dNTPs), is an early event during tumorigenesis (Kotsantis et al 2018). Such stress leads to stalling of the replisome and activation of the checkpoint kinase ATR/Mec1, which causes the subsequent activation of the effector kinase Chk1 in humans or Rad53 in yeast (Giannattasio and Branzei 2017). This response to replication stress is called the S-phase, intra-S-phase, or DNA replication checkpoint (Pardo et al 2017).…”

mentioning

confidence: 99%

“…The S-phase checkpoint results in a range of responses including the up-regulation of dNTPs, DNA repair, and fork stabilization, which enables forks to resume replication after stalling (Giannattasio and Branzei 2017). In addition, it was observed over 40 years ago that DNA damage results in the inhibition of replication initiation (Painter 1977), which is checkpoint-dependent (Painter and Young 1980).…”

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confidence: 99%

Checkpoint inhibition of origin firing prevents DNA topological stress

et al. 2019

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A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors.

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S-phase checkpoint regulations that preserve replication and chromosome integrity upon dNTP depletion

Cited by 60 publications

References 184 publications

H3K4 methylation at active genes mitigates transcription-replication conflicts during replication stress

H3K4 methylation at active genes mitigates transcription-replication conflicts during replication stress

Rad53 controls DNA unwinding after helicase-polymerase uncoupling at DNA replication forks

Checkpoint inhibition of origin firing prevents DNA topological stress

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