scholarly journals DNA repair factor RAD18 and DNA polymerase Polκ confer tolerance of oncogenic DNA replication stress

2017 ◽  
Vol 216 (10) ◽  
pp. 3097-3115 ◽  
Author(s):  
Yang Yang ◽  
Yanzhe Gao ◽  
Liz Mutter-Rottmayer ◽  
Anastasia Zlatanou ◽  
Michael Durando ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Replication Stress ◽  
Neoplastic Cells ◽  
Oncogenic Ras ◽  
Signaling Axis ◽  
Dna Replication Stress ◽  
Dna Repair Protein

The mechanisms by which neoplastic cells tolerate oncogene-induced DNA replication stress are poorly understood. Cyclin-dependent kinase 2 (CDK2) is a major mediator of oncogenic DNA replication stress. In this study, we show that CDK2-inducing stimuli (including Cyclin E overexpression, oncogenic RAS, and WEE1 inhibition) activate the DNA repair protein RAD18. CDK2-induced RAD18 activation required initiation of DNA synthesis and was repressed by p53. RAD18 and its effector, DNA polymerase κ (Polκ), sustained ongoing DNA synthesis in cells harboring elevated CDK2 activity. RAD18-deficient cells aberrantly accumulated single-stranded DNA (ssDNA) after CDK2 activation. In RAD18-depleted cells, the G2/M checkpoint was necessary to prevent mitotic entry with persistent ssDNA. Rad18−/− and Polκ−/− cells were highly sensitive to the WEE1 inhibitor MK-1775 (which simultaneously activates CDK2 and abrogates the G2/M checkpoint). Collectively, our results show that the RAD18–Polκ signaling axis allows tolerance of CDK2-mediated oncogenic stress and may allow neoplastic cells to breach tumorigenic barriers.

scholarly journals Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

2021 ◽  
Author(s):  
Cory Haluska ◽  
Fengzhi Jin ◽  
Yanchang Wang
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Protein Phosphatase ◽  
Budding Yeast ◽  
Replication Stress ◽  
S Phase ◽  
Viability Loss ◽  
Phosphatase 2A ◽  
Dna Replication Stress

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and Protein Phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2ACdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We showed that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1 that encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK ( cdc28F19) also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.

scholarly journals Stwl Modifies Chromatin Compaction and Is Required to Maintain DNA Integrity in the Presence of Perturbed DNA Replication

2009 ◽  
Vol 20 (3) ◽  
pp. 983-994 ◽  
Author(s):  
Xia Yi ◽  
Hilda I. de Vries ◽  
Katarzyna Siudeja ◽  
Anil Rana ◽  
Willy Lemstra ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Replication Stress ◽  
Epigenetic Mechanism ◽  
Dna Integrity ◽  
Silent Chromatin ◽  
Chromatin Compaction ◽  
Cycle Arrest ◽  
Dna Replication Stress

Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent accumulation of this type of DNA damage that coincides with hampered DNA synthesis. Here, we report the identification of stonewall (stwl) as a novel hydroxyurea-hypersensitive mutant. We demonstrate that Stwl is required to prevent accumulation of DNA damage induced by hydroxyurea; yet, Stwl is not involved in S/M checkpoint regulation. We show that Stwl is a heterochromatin-associated protein with transcription-repressing capacities. In stwl mutants, levels of trimethylated H3K27 and H3K9 (two hallmarks of silent chromatin) are decreased. Our data provide evidence for a Stwl-dependent epigenetic mechanism that is involved in the maintenance of the normal balance between euchromatin and heterochromatin and that is required to prevent accumulation of DNA damage in the presence of DNA replication stress.

scholarly journals Knockdown of RMI1 impairs DNA repair under DNA replication stress

2017 ◽  
Vol 494 (1-2) ◽  
pp. 158-164 ◽  
Author(s):  
Chang Xu ◽  
Lianying Fang ◽  
Yangyang Kong ◽  
Changyan Xiao ◽  
Mengmeng Yang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Replication Stress ◽  
Dna Replication Stress

scholarly journals Global analysis of genomic instability caused by DNA replication stress in Saccharomyces cerevisiae

2016 ◽  
Vol 113 (50) ◽  
pp. E8114-E8121 ◽  
Author(s):  
Dao-Qiong Zheng ◽  
Ke Zhang ◽  
Xue-Chang Wu ◽  
Piotr A. Mieczkowski ◽  
Thomas D. Petes
Keyword(s):  
Dna Replication ◽  
Genomic Instability ◽  
Dna Polymerase ◽  
Replication Stress ◽  
Dna Lesions ◽  
Chromosome Loss ◽  
Dna Breaks ◽  
Dna Polymerase Δ ◽  
Dna Replication Stress ◽  
Low Levels

DNA replication stress (DRS)-induced genomic instability is an important factor driving cancer development. To understand the mechanisms of DRS-associated genomic instability, we measured the rates of genomic alterations throughout the genome in a yeast strain with lowered expression of the replicative DNA polymerase δ. By a genetic test, we showed that most recombinogenic DNA lesions were introduced during S or G2 phase, presumably as a consequence of broken replication forks. We observed a high rate of chromosome loss, likely reflecting a reduced capacity of the low-polymerase strains to repair double-stranded DNA breaks (DSBs). We also observed a high frequency of deletion events within tandemly repeated genes such as the ribosomal RNA genes. By whole-genome sequencing, we found that low levels of DNA polymerase δ elevated mutation rates, both single-base mutations and small insertions/deletions. Finally, we showed that cells with low levels of DNA polymerase δ tended to accumulate small promoter mutations that increased the expression of this polymerase. These deletions conferred a selective growth advantage to cells, demonstrating that DRS can be one factor driving phenotypic evolution.

scholarly journals Aphidicolin does not inhibit DNA repair synthesis in ultraviolet-irradiated HeLa cells. A radioautographic study

1981 ◽  
Vol 199 (2) ◽  
pp. 453-455 ◽  
Author(s):  
N Hardt ◽  
G Pedrali-Noy ◽  
F Focher ◽  
S Spadari
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Hela Cells ◽  
Nuclear Dna ◽  
Detectable Effect ◽  
Repair Synthesis ◽  
Dna Polymerase Alpha ◽  
Dna Repair Synthesis

A radioautographic examination of nuclear DNA synthesis in unirradiated and u.v.-irradiated HeLa cells, in the presence and in the absence of aphidicolin, showed that aphidicolin inhibits nuclear DNA replication and has no detectable effect on DNA repair synthesis. Although the results establish that in u.v.-irradiated HeLa cells most of the DNA repair synthesis is not due to DNA polymerase alpha, they do not preclude a significant role for this enzyme in DNA repair processes.

scholarly journals Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress

2009 ◽  
Vol 122 (22) ◽  
pp. 4070-4080 ◽  
Author(s):  
V. M. Vassin ◽  
R. W. Anantha ◽  
E. Sokolova ◽  
S. Kanner ◽  
J. A. Borowiec
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Replication Stress ◽  
Dna Replication Stress

A mechanism for Rad53 to couple leading- and lagging-strand DNA synthesis under replication stress in budding yeast

2021 ◽  
Vol 118 (38) ◽  
pp. e2109334118
Author(s):  
Albert Serra-Cardona ◽  
Chuanhe Yu ◽  
Xinmin Zhang ◽  
Xu Hua ◽  
Yuan Yao ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Replication Stress ◽  
Replication Checkpoint ◽  
Checkpoint Pathway ◽  
Dna Replication Checkpoint ◽  
Dna Replication Stress ◽  
Leading Strand ◽  
Mutant Cells ◽  
Lagging Strand

In response to DNA replication stress, DNA replication checkpoint kinase Mec1 phosphorylates Mrc1, which in turn activates Rad53 to prevent the generation of deleterious single-stranded DNA, a process that remains poorly understood. We previously reported that lagging-strand DNA synthesis proceeds farther than leading strand in rad53-1 mutant cells defective in replication checkpoint under replication stress, resulting in the exposure of long stretches of the leading-strand templates. Here, we show that asymmetric DNA synthesis is also observed in mec1-100 and mrc1-AQ cells defective in replication checkpoint but, surprisingly, not in mrc1∆ cells in which both DNA replication and checkpoint functions of Mrc1 are missing. Furthermore, depletion of either Mrc1 or its partner, Tof1, suppresses the asymmetric DNA synthesis in rad53-1 mutant cells. Thus, the DNA replication checkpoint pathway couples leading- and lagging-strand DNA synthesis by attenuating the replication function of Mrc1-Tof1 under replication stress.

scholarly journals SAMHD1 promotes oncogene-induced replication stress

2020 ◽  
Author(s):  
Si Min Zhang ◽  
Jose M Calderón-Montaño ◽  
Sean G Rudd
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Overall Survival ◽  
Dna Replication ◽  
Replication Fork ◽  
Potential Role ◽  
Human Fibroblasts ◽  
Replication Stress ◽  
Oncogenic Ras ◽  
Dna Replication Stress

AbstractOncogenes induce DNA replication stress in cancer cells. Although this was established more than a decade ago, we are still unravelling the molecular underpinnings of this phenomenon, which will be critical if we are to exploit this knowledge to improve cancer treatment. A key mediator of oncogene-induced replication stress is the availability of DNA precursors, which will limit ongoing DNA synthesis by cellular replicases. In this study, we identify a potential role for nucleotide catabolism in promoting replication stress induced by oncogenes. Specifically, we establish that the dNTPase SAMHD1 slows DNA replication fork speeds in human fibroblasts harbouring an oncogenic RAS allele, elevating levels of endogenous DNA damage, and ultimately limiting cell proliferation. We then show that oncogenic RAS-driven tumours express reduced SAMHD1 levels, suggesting they have overcome this tumour suppressor barrier, and that this correlates with worse overall survival for these patients.Abstract Figure

scholarly journals High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing

Cell Research ◽  
2020 ◽  
Vol 30 (11) ◽  
pp. 997-1008 ◽  
Author(s):  
Morgane Macheret ◽  
Rahul Bhowmick ◽  
Katarzyna Sobkowiak ◽  
Laura Padayachy ◽  
Jonathan Mailler ◽  
...  
Keyword(s):  
Dna Replication ◽  
High Resolution ◽  
Dna Synthesis ◽  
Direct Sequencing ◽  
Replication Stress ◽  
Fragile Sites ◽  
Common Fragile Sites ◽  
Dna Replication Stress ◽  
Genomic Regions ◽  
In Cells

Abstract DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.

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