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

Mammalian polymerase theta (Polθ; encoded by POLQ ) is a unique 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-stranded break (DSB) repair. Our results show that Polθ–helicase promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells. In addition, the helicase activity suppresses CRISPR/Cas9 mediated gene targeting by homologous recombination (HR). In vitro experiments reveal that Polθ–helicase displaces RPA to facilitate the annealing of complementary DNA during alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, we show that the inhibition of RPA1 subunit enhances end-joining and suppresses recombination at telomeres. Taken together, our results reveal that the balance between HR and alt-NHEJ is regulated by opposing activities of Polθ and RPA, providing critical insight into the mechanism that control DSB repair pathway choice in mammalian cells.