Polq-Mediated End Joining Is Essential for Surviving DNA Double-Strand Breaks during Early Zebrafish Development

Thyme, Summer B.; Schier, Alexander F.

doi:10.1016/j.celrep.2016.03.072

Cited by 66 publications

(51 citation statements)

References 49 publications

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“…The clear contribution of TMEJ in genetically non-compromised embryonic stem cells agrees well with the observation that DSB-induced translocation formation in mouse embryonic cells and in mice models is mostly cNHEJ independent (Weinstock et al, 2007;Simsek & Jasin, 2010;Mateos-Gomez et al, 2015). Intriguingly, the apparent shift in end-joining pathway usage towards TMEJ in cells that have a more stem-cell like character is also found in other species: in the model systems C. elegans and zebrafish TMEJ is the dominant mode of (error-prone) DSB repair in germ and embryonic cells; somatic cells, however, employ mostly cNHEJ Thyme & Schier, 2016). In agreement, Wyatt and colleagues recently showed that mouse embryonic fibroblasts (MEFs) preferentially use cNHEJ to repair Cas9-induced blunt DSBs leading them to conclude that TMEJ functions merely as a back-up mechanism.…”

Section: Tmej Acts Parallel To Cnhej In Embryonic Cellsmentioning

confidence: 93%

Mutational signatures of non‐homologous and polymerase theta‐mediated end‐joining in embryonic stem cells

et al. 2017

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Cells employ potentially mutagenic DNA repair mechanisms to avoid the detrimental effects of chromosome breaks on cell survival. While classical non‐homologous end‐joining (cNHEJ) is largely error‐free, alternative end‐joining pathways have been described that are intrinsically mutagenic. Which end‐joining mechanisms operate in germ and embryonic cells and thus contribute to heritable mutations found in congenital diseases is, however, still largely elusive. Here, we determined the genetic requirements for the repair of CRISPR/Cas9‐induced chromosomal breaks of different configurations, and establish the mutational consequences. We find that cNHEJ and polymerase theta‐mediated end‐joining (TMEJ) act both parallel and redundant in mouse embryonic stem cells and account for virtually all end‐joining activity. Surprisingly, mutagenic repair by polymerase theta (Pol θ, encoded by the Polq gene) is most prevalent for blunt double‐strand breaks (DSBs), while cNHEJ dictates mutagenic repair of DSBs with protruding ends, in which the cNHEJ polymerases lambda and mu play minor roles. We conclude that cNHEJ‐dependent repair of DSBs with protruding ends can explain de novo formation of tandem duplications in mammalian genomes.

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Section: Tmej Acts Parallel To Cnhej In Embryonic Cellsmentioning

confidence: 93%

Mutational signatures of non‐homologous and polymerase theta‐mediated end‐joining in embryonic stem cells

et al. 2017

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“…POLQ's critical role in Alt-EJ-mediated DSB repair has been demonstrated in various animal organisms, such as Caenorhabditis elegans, flies, zebrafish, mice and human (Chan et al, 2010;Roerink et al, 2014;Kent et al, 2015;Mateos-Gomez et al, 2015;Thyme & Schier, 2016). POLQ was shown to be a key driver of CRISPR-Cas9 DSB repair leading to mutagenesis in C. elegans (van Schendel et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

POLQ plays a key role in the repair of CRISPR/Cas9‐induced double‐stranded breaks in the moss Physcomitrella patens

et al. 2019

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Summary Double‐stranded breaks can be repaired by different mechanisms such as homologous recombination (HR), classical nonhomologous end joining (C‐NHEJ) and alternative end joining (Alt‐EJ). Polymerase Q (POLQ) has been proposed to be the main factor involved in Alt‐EJ‐mediated DNA repair. Here we describe the role of POLQ in DNA repair and gene targeting in Physcomitrella patens. The disruption of the POLQ gene does not influence the genetic stability of P. patens nor its development. The polq mutant shows the same sensitivity as wild‐type towards most of the genotoxic agents tested (ultraviolet (UV), methyl methanesulfonate (MMS) and cisplatin) with the notable exception of bleomycin for which it shows less sensitivity than the wild‐type. Furthermore, we show that POLQ is involved in the repair of CRISPR‐Cas9‐induced double‐stranded breaks in P. patens. We also demonstrate that POLQ is a potential competitor and/or inhibitor of the HR repair pathway. This finding has a consequence in terms of genetic engineering, as in the absence of POLQ the frequency of gene targeting is significantly increased and the number of clean two‐sided HR‐mediated insertions is enhanced. Therefore, the control of POLQ activity in plants could be a useful strategy to optimize the tools of genome engineering for plant breeding.

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“…Alt-NHEJ was initially thought to act as a back-up mechanism 2 , however, recent studies revealed that it operates even when HR and C-NHEJ are intact 3 . Alt-NHEJ is a major repair pathway during early vertebrate development 4 . Moreover, when HR and C-NHEJ are impaired, mammalian cells become highly dependent on alt-NHEJ for survival 5–7 .…”

Section: Introductionmentioning

confidence: 99%

“…Its activity was first linked to alt-NHEJ during P-element transposition in flies 10 , and later found to promote end-joining in plants 11 , worms 12 , fish 4 , and mammals 5,6,13–15 . Mammalian Polθ stimulates alt-NHEJ in response to endonuclease-mediated cleavage of reporter constructs 5,6,13,14 , drives the fusion of dysfunctional telomeres 5 , and promotes chromosomal translocations in mouse embryonic stem (mES) cells 5 .…”

Section: Introductionmentioning

confidence: 99%

The helicase domain of Polθ counteracts RPA to promote alt-NHEJ

Mateos-Gómez

Kent

Deng

et al. 2017

Nat Struct Mol Biol

136

117

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Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative-NHEJ (alt-NHEJ). Here we perform structure-function analyses and report that, in addition to the polymerase domain, the helicase activity plays a central role during Polθ-mediated double-strand break (DSB) repair. Our results show that Polθ-helicase promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells. The helicase activity also suppresses CRISPR/Cas9 mediated gene targeting by homologous recombination (HR). In vitro experiments reveal that Polθ–helicase facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, we show that the inhibition of RPA1 enhances end-joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair pathway choice in mammalian cells.

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Polq-Mediated End Joining Is Essential for Surviving DNA Double-Strand Breaks during Early Zebrafish Development

Cited by 66 publications

References 49 publications

Mutational signatures of non‐homologous and polymerase theta‐mediated end‐joining in embryonic stem cells

Mutational signatures of non‐homologous and polymerase theta‐mediated end‐joining in embryonic stem cells

POLQ plays a key role in the repair of CRISPR/Cas9‐induced double‐stranded breaks in the moss Physcomitrella patens

The helicase domain of Polθ counteracts RPA to promote alt-NHEJ

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