IntroductionR-loops are three-stranded structures composed of an RNA-DNA hybrid and displaced single-stranded DNA (ssDNA), formed co-transcriptionally and dynamically regulated in cells1,2,3. Although physiological R-loops contribute to transcription, replication, and DNA repair, their accumulation can trigger transcription-replication conflicts, DNA damage, and genome instability1,2,3,4,5,6,7,8. Emerging work from the last decade has shown that R-loops also form at DNA double-strand breaks (DSBs), which arise either from hybridization of pre-existing transcripts or from damage-induced de novo transcription pairing with the resected ssDNA, and they modulate DNA damage response and its repair by homologous recombination (HR)9,10,11,12,13,14. HR begins with DNA-end resection at DSBs to generate 3′ ssDNA for RAD51 loading and strand invasion of the sister chromatid, enabling error-free repair15. Despite their emerging importance in HR regulation, the mechanisms governing R-loop formation, resolution, and maintenance at DSBs are still not fully understood.Mutations in BRCA2 (BReast CAncer gene 2) primarily drive breast cancer, particularly estrogen receptor-positive (ER⁺) subtypes, as well as ovarian cancers16,17. BRCA2 is essential for maintaining genomic integrity through its pivotal roles in loading RAD51 onto ssDNA during HR repair of DSBs, as well as in preventing the accumulation of ssDNA gaps and protecting nascent DNA from degradation (replication-fork protection; RFP) at stalled replication forks15,18,19,20. As BRCA2’s obligatory partner, DSS1 stabilizes BRCA2, retains it in the nucleus, and enhances RAD51 loading of RPA-coated ssDNA through its DNA-mimicking interaction with RPA15,18,19,20,21,22,23,24,25. Beyond these canonical roles, our recent work indicates that DSS1 also modulates BRCA2’s binding preference for ssDNA over dsDNA, contributing to HR, RFP, and R-loop regulation26. Despite growing evidence implicating the BRCA2-DSS1 complex in R-loop homeostasis22,26,27,28,29, how BRCA2-DSS1 promotes R-loop resolution, particularly at DSBs, and how this activity intersects with HR still remain largely undefined.To help address these knowledge gaps, we have employed proximity ligation coupled with mass spectrometry to identify BRCA2-DSS1 interactors that function in R-loop processing30,31,32. This effort has led to the identification of HELZ, an uncharacterized putative RNA helicase, as a BRCA2-DSS1 interactor. While HELZ has been previously linked to translational regulation and mRNA decay33,34, a possible role in R-loop resolution and genome maintenance has not been explored. We showed that HELZ is an ssRNA-specific R-loop resolvase essential for unwinding R-loops during transcription-coupled HR at DSBs. Furthermore, we established a critical interplay between BRCA2-DSS1 and HELZ in R-loop regulation and HR promotion, providing insights into the BRCA2 mechanisms linking R-loop homeostasis, DNA damage repair, and tumorigenesis.ResultsIdentification of HELZ as an interactor of BRCA2-DSS1We utilized proximity ligation coupled with mass spectrometry30,31,32 in an effort to capture proteins that stably or transiently interact with BRCA2-DSS1 (Supplementary Fig. 1a). First, we created various engineered biotin ligase (e.g., TurboID, Split-TurboID, BioID) tagged DSS1 constructs and found that only the BioID tag does not affect the homologous recombination function of DSS1 (Supplementary Fig. 1b). Then, we generated a stable HeLa-shDSS1 cell line expressing BioID-DSS1, where the endogenous DSS1 is depleted by doxycycline-induced shDSS1 expression26. We treated these cells with olaparib and added biotin to trigger biotinylation of potential DSS1 interaction partners, such as BRCA2 (Supplementary Fig. 1c), during the DNA damage response and repair. Finally, the biotinylated proteins were enriched using streptavidin resin and analyzed by mass spectrometry. As shown in Fig. 1a, among the top hits we identified known DSS1 interactors, such as PSMD3 and PSMD1135, confirming the specificity and sensitivity of our proximity proteomics approach. Notably, HELZ, a putative RNA helicase, also emerged as a highly ranked hit (Fig. 1a). To date, only two studies have characterized HELZ, implicating it in translational initiation and mRNA decay33,34.Fig. 1: HELZ is a BRCA2–DSS1 interactor that directly binds the BRCA2 CTRB domain.Full size imagea Volcano plots indicate HELZ as a high-ranking partner of DSS1 by BioID-LC-MS/MS. Red dots represent significantly upregulated proteins (p 1, two-sided Student’s t-test). Blue dots represent significantly downregulated proteins (p