DNA Fiber Analysis: Mind the Gap!

Quinet, Annabel; Carvajal-Maldonado, Denisse; Lemaçon, Delphine; Vindigni, Alessandro

doi:10.1016/bs.mie.2017.03.019

Cited by 169 publications

(156 citation statements)

References 65 publications

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“…The CytoK15 hi CD49f hi cells were in a quiescent state (G0/G1), whereas the CytoK15 lo CD49f lo cells contained a substantial number of S phase cells and G2/M phase cells, indicating dividing cells ( Figure 4, A and B). An enrichment of sub-bulge cells was observed in hair follicles isolated from HS patients compared with the HD group, although the difference was not statistically significant (sub-bulge 27.5% ± 16.7% vs. 19% with 2 successive pulses of the thymidine analogs IdU and CldU and monitored the progression of individual forks by DNA fiber spreading (18). We observed that ldU track length was comparable in the studied ORSCs from 3 HDs, around 5 μm ( Figure 5B).…”

Section: Resultsmentioning

confidence: 91%

Hair follicle stem cell replication stress drives IFI16/STING-dependent inflammation in hidradenitis suppurativa

Orvain¹,

Lin²,

Jean-Louis³

et al. 2020

Journal of Clinical Investigation

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Section: Resultsmentioning

confidence: 91%

Hair follicle stem cell replication stress drives IFI16/STING-dependent inflammation in hidradenitis suppurativa

Orvain¹,

Lin²,

Jean-Louis³

et al. 2020

Journal of Clinical Investigation

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“…To test this hypothesis, we performed the DNA fiber assay followed by incubation with S1 nuclease. S1 cuts at ssDNA regions and secondary DNA structures as an indicator of poor-quality DNA (Quinet et al, 2017). Indeed, labelled nascent DNA tracks were S1 sensitive in BRCA2-deficient PEO1 cells, but not in the BRCA2-proficient C4-2 cells (Figure 1B).…”

Section: Resultsmentioning

confidence: 98%

Replication gaps underlie BRCA-deficiency and therapy response

Panzarino

Krais

Peng

et al. 2019

Preprint

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Cancers that are deficient in BRCA1 or BRCA2 are hypersensitive to genotoxic agents, including platinums and other first-line chemotherapeutics. The established models propose that these cancers are hypersensitive because the chemotherapies block or degrade DNA replication forks and thereby create DNA double strand breaks, both of which require functional BRCA proteins to prevent or resolve by mechanisms termed fork protection (FP) or homologous recombination (HR). However, recent findings challenge this dogma because genotoxic agents do not initially cause DNA double strand breaks or stall replication forks. Here, we propose a new model for genotoxic chemotherapy in which ssDNA replication gaps underlie the hypersensitivity of BRCA deficient cancer, and we propose that defects in HR or FP do not. Specifically, we observed that ssDNA gaps develop in BRCA deficient cells because DNA replication is not effectively restrained in response to genotoxic stress. Moreover, we observe gap suppression (GS) by either restored fork restraint or by gap filling, both of which conferred resistance to therapy in tissue culture and BRCA patient tumors. In contrast, restored HR and FP were not sufficient to prevent hypersensitivity if ssDNA gaps were not eliminated. Together, these data suggest that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose are fundamental to the mechanism of action of genotoxic chemotherapies. Introduction:Mutations in the hereditary breast cancer genes, BRCA1 and BRCA2, first demonstrated that cancer is a genetic disease in which susceptibility to cancer could be inherited (King et al., 1986). In addition to breast cancer, mutated BRCA1 or BRCA2 also cause a predisposition to other cancer types, including breast, ovarian, pancreatic, and colorectal cancers. Importantly, cancers with mutated BRCA genes are hypersensitive to cisplatin, a first-line anti-cancer chemotherapy that has been the standard of care for ovarian cancer for over 40 years (Munnell, 1968). BRCA deficient cancers are thought to be

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“…To test the hypothesis that gaps form in the vicinity of the accelerated replication fork, following PARPi treatment, cells were incubated with the S1 nuclease that digests ssDNA regions(Quinet et al, 2017; Quinet et al, 2016). If nascent ssDNA regions are within the labelled replication tracts, S1 nuclease will cut and therefore, shorten the visible CldU replication tract.…”

Section: Resultsmentioning

confidence: 99%

PARPi synthetic lethality derives from replication-associated single-stranded DNA gaps

Cong

Kousholt

Peng

et al. 2019

Preprint

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BRCA1 or BRCA2 (BRCA)-deficient tumor cells have defects in DNA double strand break repair by homologous recombination (HR) and fork protection (FP) that are thought to underlie the sensitivity to poly(ADP-ribose) polymerase inhibitor (PARPi). Given the recent finding that PARPi accelerates DNA replication, it was proposed that high speed DNA replication leads to DNA double strand breaks (DSBs). Here, we tested the alternative hypothesis that PARPi sensitivity in BRCA deficient cells results from combined replication dysfunction that causes a lethal accumulation of replication-associated single-stranded DNA (ssDNA) gaps. In support of a gap toxicity threshold, PARPi-induced ssDNA gaps accumulate more excessively in BRCA deficient cells and are suppressed in de novo and genetic models of PARPi resistance while defects in HR or FP often lack this correlation.We also uncouple replication speed from lethality. The clear link between PARPi sensitivity and ssDNA gaps provides a new paradigm for understanding synthetic lethal interactions. Schlacher et al., 2012). Consistent with the DSB inducing model of therapy response in BRCA deficient cells, restoration of HR or FP are associated with chemoresistance (Bouwman et al., 2010; Bunting et al., 2010; Chaudhuri et al., 2016; Edwards et al., 2008; Sakai et al., 2008). However, this model of therapy response is challenged by recent reports indicating that chemotoxic agents do not initially induce DSBs or even pause DNA replication forks (Huang et al., 2013; Mutreja et al., 2018; Zellweger et al., 2015). Indeed, PARPi accelerates DNA replication (Maya-Mendoza et al., 2018). To square this finding with the framework that HR and/or FP defects drive PARPi therapy response, it was proposed that high speed DNA replication ultimately induces DSBs (Maya-Mendoza et al., 2018; Quinet and Vindigni, 2018). However, we recently proposed a competing model in which ssDNA gaps underlie the BRCA deficiency phenotype, and not DSBs and we propose are fundamental to the mechanismof-action of genotoxic therapies (Panzarino et al., 2019). 2011;Here, we provide evidence that the genotoxic lesion driving PARPi synthetic lethality in BRCA deficient cancer is wide-spread single-stranded DNA (ssDNA) gaps. Gap induction is associated with PARPi potentially due to its role in the repair of ssDNA breaks, processing Okazaki fragments, or regulating replication-fork reversal -a mechanism by which replication forks reverse direction when confronted with replication obstacles (Berti et al. Sogo et al., 2002). Critically, we demonstrate that PARPi gaps are compounded in BRCA-deficient cells, but suppressed in cell lines and tumors with intrinsic, genetic or de novo mechanisms of PARPi resistance. These findings highlight that a key function of the BRCA-RAD51 proteins is to limit replication gaps (Hashimoto et al., 2010; Kolinjivadi et al., 2017a; Kolinjivadi et al., 2017b; Zellweger et al., 2015, Panzarino et al., 2019 and that loss of this function confers therapy response. RESULTS PARPi generates s...

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DNA Fiber Analysis: Mind the Gap!

Cited by 169 publications

References 65 publications

Hair follicle stem cell replication stress drives IFI16/STING-dependent inflammation in hidradenitis suppurativa

Hair follicle stem cell replication stress drives IFI16/STING-dependent inflammation in hidradenitis suppurativa

Replication gaps underlie BRCA-deficiency and therapy response

PARPi synthetic lethality derives from replication-associated single-stranded DNA gaps

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