DNA replication stress triggers rapid DNA replication fork breakage by Artemis and XPF

Bétous, Rémy; Rugy, Théo Goullet de; Pelegrini, Alessandra Luíza; Queillé, Sophie; Villartay, Jean-Pierre de; Hoffmann, Jean-Sébastien

doi:10.1371/journal.pgen.1007541

Cited by 29 publications

(26 citation statements)

References 30 publications

(38 reference statements)

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“…The endonucleases, MUS81, ARTEMIS (DCLRE1C), and XPF-ERCC1 act on stalled replication forks to resolve DRS during the S and G2 phases and/or during mitosis and cleave singlestranded DNAs (Bétous et al, 2018). In the pre-latent phase, the transcript levels of these important enzymes vary modestly with the exception of XPF-ERCC1, which peaks on day one and two p.i.…”

Section: Does Infection With Ebv Induce a Genotoxic Signal In Primarymentioning

confidence: 99%

The first days in the life of naïve human B-lymphocytes infected with Epstein-Barr virus

Pich

Mrozek-Górska

Bouvet

et al. 2019

Preprint

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Epstein-Barr virus (EBV) infects and activates resting human B-lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cells in vitro are known, but their functions in the early, pre-latent phase of infection have not been investigated systematically. In studies during the first eight days of infection using derivatives of EBV with mutations in single genes of EBVs we found only EBNA2 to be essential for activating naïve human B-lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, LMP2A and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding EBER RNAs had no discernable phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the pre-latent phase. Even EBNA1 which has very critical longterm functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a critical parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B-lymphocytes from energetically quiescent to activated cells. Author summaryThe preferred target of Epstein-Barr virus (EBV) are human resting B-lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial 3 increase in cell volume followed by cellular DNA synthesis after three days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to three days later the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains we investigated the individual contributions of EBV's multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBV's pre-latent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B-lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming viral latent genes other than EBNA2 are dispensable but some, EBNA-LP for example, support the viral program and presumably stabilize the infected cells once viral latency is established.

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Section: Does Infection With Ebv Induce a Genotoxic Signal In Primarymentioning

confidence: 99%

The first days in the life of naïve human B-lymphocytes infected with Epstein-Barr virus

Pich

Mrozek-Górska

Bouvet

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…This DSB induction promotes replication fork restart. [ 35,36 ] Our data, showing that DNA‐PKcs is targeted to SIRs in cellular extracts, provide a possible explanation for the activation of the DNA‐PKcs that is essential for ARTEMIS endonuclease activity, when cells have to overcome a replication stress.…”

Section: Discussionmentioning

confidence: 91%

Proteomic Analysis of DNA Synthesis on a Structured DNA Template in Human Cellular Extracts: Interplay Between NHEJ and Replication‐Associated Proteins

et al. 2020

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It is established that short inverted repeats trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with structured DNA is unknown. It has been previously reported that in human cell‐free extracts, DNA primer extension using a structured single‐stranded template is transiently blocked at DNA hairpins. Here, the proteomic analysis of proteins bound to the DNA template is reported and evidence that the DNA‐PK complex (DNA‐PKcs and the Ku heterodimer) recognizes, and is activated by, structured single‐stranded DNA is provided. Hijacking the DNA‐PK complex by double‐stranded oligonucleotides results in a large removal of the pausing sites and an elevated DNA extension efficiency. Conversely, DNA‐PKcs inhibition results in its stabilization on the template, along with other proteins acting downstream in the Non‐Homologous End‐Joining (NHEJ) pathway, especially the XRCC4‐DNA ligase 4 complex and the cofactor PAXX. Retention of NHEJ factors to the DNA in the absence of DNA‐PKcs activity correlates with additional halts of primer extension, suggesting that these proteins hinder the progression of the DNA synthesis at these sites. Overall these results raise the possibility that, upon binding to hairpins formed onto ssDNA during fork progression, the DNA‐PK complex interferes with replication fork dynamics in vivo.

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“…First, recent findings suggest that DNA-PK may be dispensable for synapsis formation during NHEJ 37 . Accordingly, repair of “simple” DSBs in G1 phase may have a reduced reliance on DNA-PK, whereas repair of more “complex” DSBs in S phase may require DNA-PK, possibly in partnership with the nuclease Artemis 38–40 . Second, it is possible that DNA-PK inhibition may be more impactful in S phase due to trapping of Ku proteins at DSBs, which inhibits the activation of homologous recombination pathways 41 .…”

Section: Discussionmentioning

confidence: 99%

Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

Kumar

Chao

Roberts

et al. 2020

Preprint

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33TP53 mutations in cancer are associated with poor patient outcomes and resistance to 34 DNA damaging therapies 1-3 . However, the mechanisms underlying treatment resistance 35 in p53-deficient cells remain poorly characterized. Here, we show that p53-deficient 36 cells exhibit hyperactive repair of therapy-induced DNA double strand breaks (DSBs), 37 which is suppressed by inhibition of DNA-dependent protein kinase (DNA-PK). Single-38 cell analyses of DSB repair kinetics and cell cycle state transitions reveal an essential 39 role for DNA-PK in suppressing S phase DNA damage and mitotic catastrophe in p53-40 deficient cells. Yet, a subset of p53-deficient cells exhibit intrinsic resistance to 41 therapeutic DSBs due to a repair pathway that is not sensitive to DNA-PK inhibition. 42We show that p53 deficiency induces overexpression of DNA Polymerase Theta (Pol ), 43 which mediates an alternative end-joining repair pathway that becomes hyperactivated 44 by DNA-PK inhibition 4 . Combined inhibition of DNA-PK and Pol  restores therapeutic 45 DNA damage sensitivity in p53-deficient cells. Thus, our study identifies two targetable 46 DSB end joining pathways that can be suppressed as a strategy to overcome resistance 47The mechanisms for therapeutic resistance in p53-deficient cells remains poorly 57 characterized. Past work has suggested a role for loss of p53-mediated apoptosis 12,15 . 58However, the response of epithelial cancer cells to DNA damaging therapy is often 59 determined by the efficiency of inducing senescence or mitotic catastrophe, rather than 60 apoptosis 16,17 . p53 is also a transcription factor that responds to DNA double strand 61 breaks (DSBs) to determine cellular fate 7,18 . Recent insights have revealed the 62 importance of p53-signaling waves in regulation of cellular fate decisions of quiescence 63 versus cell cycle re-entry after DNA damage 19,20 . However, the mechanisms that 64 determine such cell fate decisions upon DNA damage induction in p53-mutant epithelial 65 cells have not been established, and may lead to novel strategies to restore treatment 66 sensitivity. 67In this study, we investigate altered DNA repair mechanisms in p53 deficiency as 68 a major contributor to resistance to DNA damaging therapies. We find that p53-deficient 69 cells exhibit hyperactive repair and accelerated resolution of DNA damage foci. Utilizing 70 live-cell imaging, we show that this ability to resolve DNA damage rapidly is partially 71 dependent on DNA-PK, a critical serine/threonine kinase in the non-homologous end 72 joining (NHEJ) pathway 21 . Inhibition of DNA-PK using the small molecule inhibitor 73 NU7441 partially sensitizes p53-deficient cells to DSB inducing agents. We further show 74 that this effect is specifically due to propagation of S phase related damage leading to 75 mitotic catastrophe, highlighting a role for DNA-PK in S phase DNA damage repair that 76 was previously under appreciated. Furthermore, using chromosomal break repair 77 assays we show that in the context of inhibitor t...

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DNA replication stress triggers rapid DNA replication fork breakage by Artemis and XPF

Cited by 29 publications

References 30 publications

The first days in the life of naïve human B-lymphocytes infected with Epstein-Barr virus

The first days in the life of naïve human B-lymphocytes infected with Epstein-Barr virus

Proteomic Analysis of DNA Synthesis on a Structured DNA Template in Human Cellular Extracts: Interplay Between NHEJ and Replication‐Associated Proteins

Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

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