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

Anjali Mann, Miguel Angel Ramirez-Otero, Anna De Antoni, Yodhara Wijesekara Hanthi, Vincenzo Sannino, Giorgio Baldi, Lucia Falbo, Anna Schrempf, Sara Bernardo, Joanna Loizou, Vincenzo Costanzo

Molecular cell 2022 Nov 17

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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. Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

Citation

Anjali Mann, Miguel Angel Ramirez-Otero, Anna De Antoni, Yodhara Wijesekara Hanthi, Vincenzo Sannino, Giorgio Baldi, Lucia Falbo, Anna Schrempf, Sara Bernardo, Joanna Loizou, Vincenzo Costanzo. POLθ prevents MRE11-NBS1-CtIP-dependent fork breakage in the absence of BRCA2/RAD51 by filling lagging-strand gaps. Molecular cell. 2022 Nov 17;82(22):4218-4231.e8