Microhomology-mediated end joining acts directly on replication forks to repair single-ended double-strand breaks.

Replication stress, intrinsic to oncogenesis, often leads to fork breakage and double-strand break (DSB) formation. Conventionally, break-induced replication (BIR) is considered the primary mechanism for repairing replication-associated single-ended DSBs (seDSBs). Here, we demonstrate that microhomology-mediated end joining (MMEJ) acts directly to repair seDSBs at broken replication forks (fork-MMEJ), preferentially on the leading strands, and functions cooperatively with BIR. While promoted by DNA polymerase theta (Polθ), fork-MMEJ operates independently of MRE11/CtIP-mediated end resection, relies on RPA, and produces asymmetric deletion patterns, distinct from canonical MMEJ (cMMEJ), which is defined at replication-independent double-ended DSBs (deDSBs). ATR, activated as end resection proceeds, serves as a pivotal switch to suppress fork-MMEJ while promoting BIR. The combined inactivation of ATR and Polθ synergistically kills cancer cells under high replication stress with minimal toxicity to normal cells. Together, our study provides fundamental insights into the MMEJ mechanism and offers new strategies for cancer treatment.