Spontaneous slow replication fork progression elicits mitosis alterations in homologous recombination-deficient mammalian cells

Wilhelm, Thérèse; Magdalou, Indiana; Barascu, Aurélia; Técher, Hervé; Debatisse, Michèlle; Lopez, Bernard S.

doi:10.1073/pnas.1311520111

Cited by 75 publications

(100 citation statements)

References 63 publications

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“…The contribution of Rad51 to unperturbed replication was more evident in the hamster cell line V79Puro expressing a dominantnegative Rad51 mutant (V79Puro/SM24) [ Fig. S4G and previous reports (30,31)]. Surprisingly, after Rad51 knockdown, UV irradiation caused a significant shortening of the CldU track synthesized before UV irradiation (Fig.…”

Section: Resultssupporting

confidence: 60%

“…U2OS and HeLa cancer cells, with intact and disrupted p53 pathways, respectively (American Type Culture Collection); GM0637 and XPA-deficient (GM04312) fibroblasts transformed with SV40 LT antigen (Coriell Repository); and V79 and SM24 cells [gifts from B. Lopez, Institut Gustave Roussy, Villejuif, France (30,31)] were grown in DMEM (Invitrogen) with 10% (vol/vol) FCS. Transfections were performed using Jet Prime (Polyplus).…”

Section: Methodsmentioning

confidence: 99%

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Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

Vallerga

Mansilla

Federico

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UVdamaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.PrimPol | polκ | polη | DNA damage tolerance | DNA replication T he DNA-binding protein Rad51 is a central component of homologous recombination repair (HRR). HRR repairs doublestrand breaks (DSBs) in an error-free way and processes one-ended DSBs to reactivate collapsed replication forks (1). During HRR, DSBs are processed by the 3′-to-5′ exonuclease activity of the double strand break repair nuclease (Mre11) to generate protruding 3′ ssDNA at DSBs. The ssDNA is then coated with Rad51, a factor that catalyzes homology search and strand invasion. The loading and stabilization of Rad51/ssDNA complexes are supported by multiple mediators, such as the tumor suppressor BRCA2 (breast cancer 2) (1). Moreover, Rad51 promotes XPF1-and Exo1-mediated DSB formation after gemcitabine-induced irreversible ribonucleotide reductase inhibition, thus promoting cell death (2). The signals that redirect Rad51 into a DSB formation pathway rather than DSB repair are not yet known.The functions of Rad51 are not limited to the processing/ generation of DSBs. Over the past few years, it has become evident that Rad51 escorts ongoing replication forks regardless of the presence of DSBs (3-5). Specifically, Rad51 protects persistently stalled replication forks from Mre11-mediated nucleolytic degradation and facilitates replication fork restart when the replication-halting agent hydroxyurea (HU) or aphidicolin (APH) is removed (6-19). Such novel functions of Rad51 require many HRR factors, including BCRA2, FANCD2 (Fanconi Anemia Complementation group protein D2), CtIP, BRCA1, and the WRN helicase, but are independent of HRR effectors, such as Rad54 (6, 7). Rad51-dependent fork-restart and fork-protection ...

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Section: Resultssupporting

confidence: 60%

Section: Methodsmentioning

confidence: 99%

Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

Vallerga

Mansilla

Federico

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…This type of mild chronic replication stress has been reported to remain undetected by cell cycle checkpoints thus becoming a serious threat to chromosomal integrity and genomic stability. 26 Altogether these data suggest that DEK promotes proliferation in particular under conditions of mild replication stress helping cells to overcome a cell cycle delay in G2/M and proceed to the next round of cell division.…”

Section: Resultsmentioning

confidence: 82%

The human oncoprotein and chromatin architectural factor DEK counteracts DNA replication stress

et al. 2014

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DNA replication stress is a major source of DNA strand breaks and genomic instability, and a hallmark of precancerous lesions. In these hyperproliferative tissues, activation of the DNA damage response results in apoptosis or senescence preventing or delaying their development to full malignancy. In cells, in which this antitumor barrier is disabled by mutations (for example, in p53), viability and further uncontrolled proliferation depend on factors that help to cope with replication-associated DNA damage. Replication problems preferentially arise in chromatin regions harboring complex DNA structures. DEK is a unique chromatin architectural factor which binds to non-B-form DNA structures, such as cruciform DNA or four-way junctions. It regulates DNA topology and chromatin organization, and is essential for the maintenance of heterochromatin integrity. Since its isolation as part of an oncogenic fusion in a subtype of AML, DEK has been consistently associated with tumor progression and chemoresistance. How DEK promotes cancer, however, is poorly understood. Here we show that DEK facilitates cellular proliferation under conditions of DNA replication stress by promoting replication fork progression. DEK also protects from the transmission of DNA damage to the daughter cell generation. We propose that DEK counteracts replication stress and ensures proliferative advantage by resolving problematic DNA and/or chromatin structures at the replication fork. INTRODUC110NDEK is a biochemically and structurally unique non-histone chromatin protein which is conserved in all higher eukaryotes. It binds to non-8-form DNA structures, such as cruciform DNA or four-way junctions, regulates DNA topology and chromatin organization, and is essential for the maintenance of heterochromatin integrity. •2 At the cellular level, it displays pleiotropic functions and has been shown to influence differentiation, apoptosis, senescence and maintenance of cell stemness. -6Since its isolation as part of an oncogenic fusion in a subtype of acute myeloid leukemia, 7 DEK has been consistently associated with tumor progression and chemoresistance.s-10 Several lines of evidence, such as DEK-dependent formation of papilloma in a mouse model of skin cancerogenesis, 10 have led to its classification as a bona fide oncogene. In melanomas, high levels of DEK expression correlate positively with metastatic potential. chemoresistance and poor treatment outcome. Significantly, DEK expression levels can distinguish benign nevi from malignant melanoma, raising the possibility of using DEK as a tumor marker. • 9We and others have shown that DEK is involved in DNA repair and the response to DNA damage: cells with downregulated DEK expression are hypersensitive to genotoxic insults and show increased susceptibility to sublethal doses of DNA damaging agents. •12 DNA repair is affected by DEK ablation, as DNA strand breaks (DSBs) are repaired less efficiently and nonhomologous end-joining appears to be defective. 12 · 13 DEK is also a substrate for covalent and n...

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“…HU is used for cell cycle synchronization [269], replication fork stability studies [249,252], studies of recovery mechanisms after the release of RS [242] and checkpoint responses [241]. Lower concentrations are used for RS induction [254], induction of senescence [74], apoptosis [257], and repair pathways induction [217]. HU reaches plasma concentrations around 0.1 mM; this should be bear in mind when interpreting the data for clinical relevance [261].…”

Section: Compoundsmentioning

confidence: 99%

Common Chemical Inductors of Replication Stress: Focus on Cell‐Based Studies

et al. 2017

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DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses.

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Spontaneous slow replication fork progression elicits mitosis alterations in homologous recombination-deficient mammalian cells

Cited by 75 publications

References 63 publications

Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

The human oncoprotein and chromatin architectural factor DEK counteracts DNA replication stress

Common Chemical Inductors of Replication Stress: Focus on Cell‐Based Studies

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