The SMC5/SMC6 complex is critical for resolving R-loop-induced transcription-replication conflicts.

R-loops play essential physiological roles but also pose a significant threat to genome stability, particularly during replication, by exacerbating transcription-replication conflicts (TRCs). In this study, we have uncovered a critical role of the SMC5/6 complex in resolving TRCs to preserve fork integrity. We identified the SMC5/6 complex as a synthetic lethal partner of senataxin (SETX), an RNA/DNA helicase critical for removing R-loops that arise during replication. We demonstrated that in SETX-deficient cells, the SMC5/6 complex is recruited to TRCs in response to the buildup of DNA supercoiling and facilitates the recruitment of the BLM/TOP3A/RMI1/RMI2 complex (BTRR). Once recruited, BTRR acts to resolve the TRCs in a manner dependent on the catalytic activity of TOP3A. BTRR is also required for FANCM accumulation at TRCs, which activates the FANCD2 pathway to resolve TRCs. These studies underscore the role of SMC5/6 in sensing TRCs and define the SMC5/6-BTRR-FANCM-FANCD2 axis as an important player in mitigating TRC-induced genome instability. Our findings also provide therapeutic opportunities for targeting this axis for effective treatment of SETX-deficient tumors.