IntroductionR loops are nucleic acids structures consisting of an RNA-DNA hybrid with a displaced single-stranded DNA (ssDNA). R loops are prevalent, occupying about 5% of the human genome1, and can form in multiple contexts, most frequently when a nascent RNA transcript hybridizes with the template ssDNA2,3. R loops are involved in diverse cellular processes, including transcription, DNA replication, DNA repair, telomere homeostasis, histone modification, and immunoglobulin class switch recombination2,3,4,5,6,7,8. While R loops play an important role in many physiological functions, dysregulation of R loop homeostasis resulting in unresolved or excessive accumulation of R loops can lead to DNA damage and genome instability2,3,4,5,6,7,8. Dysregulation of R loop homeostasis is associated with several diseases, including autoimmune disease, neurological disorders, and cancer9. Thus, the formation, resolution, and prevention of R loops must be tightly regulated.DNA double-strand breaks (DSB) are highly cytotoxic lesions which must be repaired in order to preserve genome integrity10,11,12. DSBs are repaired predominantly by two major pathways: homologous recombination (HR), which involves repair using a sister chromatid as a template and is error-free, and non-homologous end joining (NHEJ), which involves ligation of broken DNA ends and is error-prone. HR is initiated by resection of broken DNA ends to generate short 3’ ssDNA overhangs, which is facilitated by the BRCA1 breast tumor suppressor protein13,14,15,16,17,18. R loops and/or RNA-DNA hybrids have been reported at DSB sites19,20,21,22,23,24,25 and can both promote20,22,23,24 and impair DSB repair21,26,27,28,29,30 in a context-dependent manner. However, the precise mechanisms by which R loops are resolved to promote DSB repair are not well understood.Helicase with Zinc Finger (HELZ) is a member of the superfamily I (SF1) class of RNA helicases that alter the conformation of RNA by unwinding double-stranded regions. Despite HELZ being cloned over 30 years ago, very little is known about its function with only two reported HELZ-focused manuscripts indicating a helicase-independent role in protein translation and mRNA stability31,32. HELZ promotes cell proliferation, translation initiation, and ribosomal protein 6 phosphorylation32 and interacts with the carbon catabolite repressor 4-negative on TATA box (CCR4-NOT) deadenylase complex to cause decay of bound mRNAs31. HELZ is evolutionarily conserved in metazoa, and its paralogs include SETX, AQR, DNA2, and UPF1, which have been implicated in DNA repair and/or R loop regulation26,33,34,35,36. HELZ contains an amino-terminal C3H1-type zinc finger motif, a Walker A motif conferring ATP binding, a Walker B DEAA [Asp, Glu, Ala, Ala] box helicase motif, and an unstructured carboxyl-terminal region with a conserved polyA binding protein interacting motif 2 (PAM2); however, HELZ’s biochemical activity as a helicase has not been established. Moreover, HELZ has not previously been characterized in regulating R loops or DNA repair.In this work, we identify HELZ as a regulator of R loop resolution that facilitates HR-mediated DSB repair. We further demonstrate that HELZ promotes BRCA1 recruitment, DNA-end resection, and HR, thereby maintaining genome stability and conferring resistance to DSB-inducing agents.ResultsA synthetic lethal siRNA screen targeting nuclear enzymes identifies HELZ as a mediator of etoposide resistanceTo identify genes critical for governing resistance to etoposide, a topoisomerase II inhibitor and chemotherapeutic drug, we performed an etoposide sensitivity screen with a siRNA library targeting 1006 annotated nuclear enzymes in H128 small cell lung cancer (SCLC) cells, derived from a treatment-refractory tumor (Fig. 1a). Sensitivity results from the screen are shown as a plot of −Log10 (p-value) against -strictly standardized mean difference (SSMD) (Fig. 1b). 61 etoposide resistance genes were identified using the following criteria: an average cell viability