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Nat Struct Mol Biol. 2017 Dec;24(12):1116-1123. doi: 10.1038/nsmb.3494. Epub 2017 Oct 23.

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

Author information

1
Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, USA.
2
Department of Cell Biology, New York University School of Medicine, New York, New York, USA.
3
Temple University, Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA.

Abstract

Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity 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, 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.

PMID:
29058711
PMCID:
PMC6047744
DOI:
10.1038/nsmb.3494
[Indexed for MEDLINE]
Free PMC Article

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