POLθ prevents MRE11-NBS1-CtIP-dependent fork breakage in the absence of BRCA2/RAD51 by filling lagging-strand gaps

POLθ prevents MRE11-NBS1-CtIP-dependent fork breakage in the absence of BRCA2/RAD51 by filling lagging-strand gaps

POLθ promotes repair of DNA double-strand breaks (DSBs) resulting from collapsed forks in homologous recombination (HR) defective tumors. Inactivation of POLθ results in synthetic lethality with the loss of HR genes BRCA1/2, which induces under-replicated DNA accumulation. However, it is unclear whe...

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Published in:Molecular cell Vol. 82; no. 22; pp. 4218 - 4231.e8
Main Authors: Mann, Anjali, Ramirez-Otero, Miguel Angel, De Antoni, Anna, Hanthi, Yodhara Wijesekara, Sannino, Vincenzo, Baldi, Giorgio, Falbo, Lucia, Schrempf, Anna, Bernardo, Sara, Loizou, Joanna, Costanzo, Vincenzo
Format: Journal Article
Language:English
Published: United States Elsevier Inc 17-11-2022
Subjects:
BRCA2/RAD51
DNA
DNA Replication
DNA replication stress
DNA-Binding Proteins - genetics
fork breakage
Homologous Recombination - genetics
MRE11 Homologue Protein - genetics
MRE11 Homologue Protein - metabolism
MRE11-NBS1-CtIP
POL theta
ssDNA gaps
Xenopus laevis
fork breakage
Xenopus laevis
MRE11-NBS1-CtIP
BRCA2/RAD51
ssDNA gaps
POL theta
DNA replication stress
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Summary:POLθ promotes repair of DNA double-strand breaks (DSBs) resulting from collapsed forks in homologous recombination (HR) defective tumors. Inactivation of POLθ results in synthetic lethality with the loss of HR genes BRCA1/2, which induces under-replicated DNA accumulation. However, it is unclear whether POLθ-dependent DNA replication prevents HR-deficiency-associated lethality. Here, we isolated Xenopus laevis POLθ and showed that it processes stalled Okazaki fragments, directly visualized by electron microscopy, thereby suppressing ssDNA gaps accumulating on lagging strands in the absence of RAD51 and preventing fork reversal. Inhibition of POLθ DNA polymerase activity leaves fork gaps unprotected, enabling their cleavage by the MRE11-NBS1-CtIP endonuclease, which produces broken forks with asymmetric single-ended DSBs, hampering BRCA2-defective cell survival. These results reveal a POLθ-dependent genome protection function preventing stalled forks rupture and highlight possible resistance mechanisms to POLθ inhibitors. [Display omitted] •POLθ is recruited onto stalled replication forks preventing ssDNA gaps and breakage•POLθ polymerase extends stalled OKFs, whereas POLθ helicase removes them•POLθ complements the absence of BRCA2/RAD51 at replication forks•POLθ prevents MRE11-NBS1-CtIP endonuclease-dependent fork breakage In this study, Mann et al. discover that POLθ protects the vertebrate genome from the accumulation of DNA gaps, which can lead to chromosome breakage in the absence of BRCA2/RAD51. This novel function of POLθ promotes BRCA2-defective cancer cell survival and can be targeted with a specific inhibitor.
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ISSN:1097-2765
1097-4164
DOI:10.1016/j.molcel.2022.09.013