DNA replication stress induced by trifluridine determines tumor cell fate according to p53 status

ABSTRACTDNA replication stress is a predominant cause of genome instability, a driver of tumorigenesis and malignant progression. Nucleoside analog-type chemotherapeutic drugs introduce DNA damage and exacerbate DNA replication stress in tumor cells. However, the mechanisms underlying tumor cytotoxicity triggered by the drugs are not fully understood. Here, we show that the fluorinated thymidine analog trifluridine (FTD), an active component of the chemotherapeutic drug trifluridine/tipiracil, delayed DNA synthesis by human replicative DNA polymerases. FTD acted as an inefficient deoxyribonucleotide triphosphate source (FTD triphosphate) and as an obstacle base (trifluorothymine) in the template DNA strand. At the cellular level, FTD decreased thymidine triphosphate in the dNTP pool and induced FTD triphosphate accumulation, resulting in replication fork stalling caused by FTD incorporation into DNA. DNA lesions involving single-stranded DNA were generated as a result of replication fork stalling, and the p53-p21 pathway was activated. Although FTD suppressed tumor cell growth irrespective of p53 status, tumor cell fate diverged at the G2/M phase transition according to p53 status; tumor cells with wild-type p53 underwent cellular senescence via mitosis skip, whereas tumor cells that lost wild-type p53 underwent apoptotic cell death via aberrant late mitosis with severely impaired separation of sister chromatids. These results suggest that DNA replication stress induced by a nucleoside analog-type chemotherapeutic drug triggers tumor cytotoxicity by determining tumor cell fate according to p53 status.SignificanceThis study identified a unique type of DNA replication stress induced by trifluridine, which directs tumor cell fate either toward cellular senescence or apoptotic cell death according to p53 status.

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DNA Replication Stress Induced by Trifluridine Determines Tumor Cell Fate According to p53 Status

Molecular Cancer Research ◽

10.1158/1541-7786.mcr-19-1051 ◽

2020 ◽

Vol 18 (9) ◽

pp. 1354-1366 ◽

Cited By ~ 1

Author(s):

Yuki Kataoka ◽

Makoto Iimori ◽

Ryo Fujisawa ◽

Tomomi Morikawa-Ichinose ◽

Shinichiro Niimi ◽

...

Keyword(s):

Dna Replication ◽

Tumor Cell ◽

Cell Fate ◽

Replication Stress ◽

Dna Replication Stress ◽

P53 Status

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Chromatin as a Platform for Modulating the Replication Stress Response

Genes ◽

10.3390/genes9120622 ◽

2018 ◽

Vol 9 (12) ◽

pp. 622 ◽

Cited By ~ 6

Author(s):

Louis-Alexandre Fournier ◽

Arun Kumar ◽

Peter Stirling

Keyword(s):

Dna Replication ◽

Stress Response ◽

Replication Fork ◽

Replication Stress ◽

Genome Maintenance ◽

Post Translational Modifications ◽

Nucleosome Dynamics ◽

Dna Replication Stress ◽

Eukaryotic Dna ◽

Fork Stalling

Eukaryotic DNA replication occurs in the context of chromatin. Recent years have seen major advances in our understanding of histone supply, histone recycling and nascent histone incorporation during replication. Furthermore, much is now known about the roles of histone remodellers and post-translational modifications in replication. It has also become clear that nucleosome dynamics during replication play critical roles in genome maintenance and that chromatin modifiers are important for preventing DNA replication stress. An understanding of how cells deploy specific nucleosome modifiers, chaperones and remodellers directly at sites of replication fork stalling has been building more slowly. Here we will specifically discuss recent advances in understanding how chromatin composition contribute to replication fork stability and restart.

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Targeting the DNA replication stress phenotype of KRAS mutant cancer cells

Scientific Reports ◽

10.1038/s41598-021-83142-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tara Al Zubaidi ◽

O. H. Fiete Gehrisch ◽

Marie-Michelle Genois ◽

Qi Liu ◽

Shan Lu ◽

...

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Replication Stress ◽

Proton Radiation ◽

Wild Type ◽

Cellular Effects ◽

Proton Treatment ◽

Dna Replication Stress ◽

Fork Stalling ◽

Mutant Cells

AbstractMutant KRAS is a common tumor driver and frequently confers resistance to anti-cancer treatments such as radiation. DNA replication stress in these tumors may constitute a therapeutic liability but is poorly understood. Here, using single-molecule DNA fiber analysis, we first characterized baseline replication stress in a panel of unperturbed isogenic and non-isogenic cancer cell lines. Correlating with the observed enhanced replication stress we found increased levels of cytosolic double-stranded DNA in KRAS mutant compared to wild-type cells. Yet, despite this phenotype replication stress-inducing agents failed to selectively impact KRAS mutant cells, which were protected by CHK1. Similarly, most exogenous stressors studied did not differentially augment cytosolic DNA accumulation in KRAS mutant compared to wild-type cells. However, we found that proton radiation was able to slow fork progression and preferentially induce fork stalling in KRAS mutant cells. Proton treatment also partly reversed the radioresistance associated with mutant KRAS. The cellular effects of protons in the presence of KRAS mutation clearly contrasted that of other drugs affecting replication, highlighting the unique nature of the underlying DNA damage caused by protons. Taken together, our findings provide insight into the replication stress response associated with mutated KRAS, which may ultimately yield novel therapeutic opportunities.

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MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

10.1101/472654 ◽

2018 ◽

Author(s):

Emily Yun-chia Chang ◽

James P. Wells ◽

Shu-Huei Tsai ◽

Yan Coulombe ◽

Yujia A. Chan ◽

...

Keyword(s):

Dna Replication ◽

Fanconi Anemia ◽

Replication Fork ◽

Genome Instability ◽

Human Cancer ◽

Replication Stress ◽

Maintenance Factors ◽

Genome Wide ◽

A Genome ◽

Dna Replication Stress

SUMMARYEctopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors such as RAD50. We show in yeast and human cells that R-loops accumulate during RAD50 depletion. In human cancer cell models, we find that RAD50 and its partners in the MRE11-RAD50-NBS1 complex regulate R-loop-associated DNA damage and replication stress. We show that a non-nucleolytic function of MRE11 is important for R-loop suppression via activation of PCNA-ubiquitination by RAD18 and recruiting anti-R-loop helicases in the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms of transcription-replication conflicts.

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Faculty Opinions recommendation of Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014466.193566 ◽

2003 ◽

Author(s):

Frederick R Cross

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Replication Stress ◽

Dna Replication Stress

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Targeting KDM4A epigenetically activates tumor-cell-intrinsic immunity by inducing DNA replication stress

Molecular Cell ◽

10.1016/j.molcel.2021.02.038 ◽

2021 ◽

Author(s):

Wuchang Zhang ◽

Wei Liu ◽

Lingfei Jia ◽

Demeng Chen ◽

Insoon Chang ◽

...

Keyword(s):

Dna Replication ◽

Tumor Cell ◽

Replication Stress ◽

Intrinsic Immunity ◽

Dna Replication Stress

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Chk1 and p21 Cooperate to Prevent Apoptosis during DNA Replication Fork Stress

Molecular Biology of the Cell ◽

10.1091/mbc.e05-07-0594 ◽

2006 ◽

Vol 17 (1) ◽

pp. 402-412 ◽

Cited By ~ 126

Author(s):

Rene Rodriguez ◽

Mark Meuth

Keyword(s):

Dna Replication ◽

Cellular Response ◽

Replication Fork ◽

Replication Stress ◽

S Phase ◽

Cell Cycle Checkpoints ◽

Primary Role ◽

Excess Thymidine ◽

Dna Replication Stress ◽

Interfering Rna

Cells respond to DNA replication stress by triggering cell cycle checkpoints, repair, or death. To understand the role of the DNA damage response pathways in determining whether cells survive replication stress or become committed to death, we examined the effect of loss of these pathways on cellular response to agents that slow or arrest DNA synthesis. We show that replication inhibitors such as excess thymidine, hydroxyurea, and camptothecin are normally poor inducers of apoptosis. However, these agents become potent inducers of death in S-phase cells upon small interfering RNA-mediated depletion of the checkpoint kinase Chk1. This death response is independent of p53 and Chk2. p21-deficient cells, on the other hand, produce a more robust apoptotic response upon Chk1 depletion. p21 is normally induced only late after thymidine treatment. In Chk1-depleted cells p21 induction occurs earlier and does not require p53. Thus, Chk1 plays a primary role in the protection of cells from death induced by replication fork stress, whereas p21 mediates through its role in regulating entry into S phase. These findings are of potential importance to cancer therapy because we demonstrate that the efficacy of clinically relevant agents can be enhanced by manipulation of these signaling pathways.

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RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse

Frontiers in Genetics ◽

10.3389/fgene.2021.753535 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nathan Ellis ◽

Jianmei Zhu ◽

Mary K Yagle ◽

Wei-Chih Yang ◽

Jing Huang ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Replication Stress ◽

Dna Helicase ◽

Replication Forks ◽

Replication Origins ◽

Dna Double Strand Breaks ◽

Response Factors ◽

Fork Stalling ◽

In Cells

Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress. Contrary to its role in repair of γ-irradiation-induced DNA double-strand breaks (DSBs), our analysis revealed that RNF4 does not significantly impact recognition or repair of replication stress-associated DSBs. Rather, using DNA fiber assays, we found that the firing of new DNA replication origins, which is required for replication restart following prolonged stress, was inhibited in cells depleted of RNF4. We also provided evidence that RNF4 recognizes and ubiquitylates sumoylated Bloom syndrome DNA helicase BLM and thereby promotes its proteosome-mediated turnover at damaged DNA replication forks. Consistent with it being a functionally important RNF4 substrate, co-depletion of BLM rescued defects in the firing of new replication origins observed in cells depleted of RNF4 alone. We concluded that RNF4 acts to remove sumoylated BLM from collapsed DNA replication forks, which is required to facilitate normal resumption of DNA synthesis after prolonged replication fork stalling and collapse.

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