PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

Piberger, Ann Liza; Bowry, Akhil; Kelly, Richard; Walker, Alexandra K.; González, Daniel; Bailey, Laura J.; Doherty, Aidan J.; Méndez, Juan Pablo; Morris, Joanna R.; Bryant, Helen E.; Petermann, Eva

doi:10.1101/773242

Cited by 11 publications

(11 citation statements)

References 61 publications

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“…Previous studies showed that FANCD2 can protect nascent DNA from degradation upon the replication stress. However, the major question is how FANCD2 protects the stalled fork and what structure it is being acted upon (Petermann et al, 2010; Piberger et al, 2019). To investigate this question, we first verified over-resection in FANCD2 deficient cells with RPA2 phosphorylation as a surrogate for measuring ssDNA arising during resection in the presence of HU.…”

Section: Resultsmentioning

confidence: 99%

FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability

Liu

Roubal

Polaczek

et al. 2021

Preprint

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SummaryFANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea induced stalled forks. The mechanisms of the fork protection are not well studied. Here, we purified FANCD2 to study how FANCD2 regulates DNA resection at stalled forks. In vitro, we showed that FANCD2 inhibits fork degradation in two ways: 1) it inhibits DNA2 nuclease activity by directly binding to DNA2. 2) independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit various nucleases, including DNA2. More unexpectedly, FANCD2 acts as a RAD51 mediator to stimulate the strand exchange activity of RAD51, and does so by enhancing ssDNA binding of RAD51. Our work biochemically explains mechanisms by which FANCD2 protects stalled forks and further provides a simple molecular explanation for genetic interactions between FANCD2 and the BRCA2 mediator.

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Section: Resultsmentioning

confidence: 99%

FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability

Liu

Roubal

Polaczek

et al. 2021

Preprint

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“…This effect could be entirely attributed to PrimPol The ICL-localized DNA fiber assay can be combined with S1 nuclease, which cleaves at ssDNA gaps and has been used to evaluate repriming events in other contexts (e.g. Bai et al, 2020;Piberger et al, 2020;Quinet et al, 2020). As expected if ssDNA was generated after the lesion, S1 eliminated many IdU tracks downstream of ICLs, resulting in an apparent decrease of traverse reactions and a concomitant increase in the frequency of apparently stalled forks (Fig 3D and E).…”

Section: Primpol Engages In Repriming Reactions At Iclsmentioning

confidence: 99%

PrimPol‐mediated repriming facilitates replication traverse of DNA interstrand crosslinks

et al. 2021

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DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Pola/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.

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“…PRIMPOL promotes DNA repriming downstream of a lesion or replication block to facilitate fork restart following ultraviolet (UV) damage and is important for interstrand cross-link traverse (3)(4)(5)(6)(7). Other studies indicate that PRIMPOL can act as a TLS polymerase in the presence of bulky adducts, prevents RNA-DNA hybrid (R-loop) formation, and is important for tolerance of G4quadruplex accumulation by repriming downstream from this obstacle (8)(9)(10).…”

Section: Introductionmentioning

confidence: 99%

CHK1 phosphorylates PRIMPOL to promote replication stress tolerance

et al. 2022

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Replication-coupled DNA repair and damage tolerance mechanisms overcome replication stress challenges and complete DNA synthesis. These pathways include fork reversal, translesion synthesis, and repriming by specialized polymerases such as PRIMPOL. Here, we investigated how these pathways are used and regulated in response to varying replication stresses. Blocking lagging-strand priming using a POLα inhibitor slows both leading- and lagging-strand synthesis due in part to RAD51-, HLTF-, and ZRANB3-mediated, but SMARCAL1-independent, fork reversal. ATR is activated, but CHK1 signaling is dampened compared to stalling both the leading and lagging strands with hydroxyurea. Increasing CHK1 activation by overexpressing CLASPIN in POLα-inhibited cells promotes replication elongation through PRIMPOL-dependent repriming. CHK1 phosphorylates PRIMPOL to promote repriming irrespective of the type of replication stress, and this phosphorylation is important for cellular resistance to DNA damage. However, PRIMPOL activation comes at the expense of single-strand gap formation, and constitutive PRIMPOL activity results in reduced cell fitness.

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PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

Cited by 11 publications

References 61 publications

FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability

FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability

PrimPol‐mediated repriming facilitates replication traverse of DNA interstrand crosslinks

CHK1 phosphorylates PRIMPOL to promote replication stress tolerance

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