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. We also showed that while fork-MMEJ is promoted by Polθ, it operates independently of MRE11/CtIP-mediated end resection, relies on RPA, and produces asymmetric deletion patterns, which is distinct from canonical MMEJ (cMMEJ) 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. 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.
Authors
- Anagh Ray (ORCID: https://orcid.org/0000-0002-2380-6467)
- David M. Gilbert (ORCID: https://orcid.org/0000-0001-8087-9737)
- Sameer Bikram Shah (ORCID: https://orcid.org/0000-0001-9442-0054)
- Takayo Sasaki (ORCID: https://orcid.org/0000-0001-7609-9316)
- Salvatore Loguercio (ORCID: https://orcid.org/0000-0002-7544-2992)
- Youhang Li (ORCID: https://orcid.org/0009-0003-8128-0154)
- Zi Wang (ORCID: https://orcid.org/0000-0002-3881-2544)
- Yuqin Zhao (ORCID: https://orcid.org/0009-0000-1656-110X)
- Shibo Li (ORCID: https://orcid.org/0000-0002-0147-8618)
- Tran Nguyen (ORCID: https://orcid.org/0000-0002-7714-1034)
- Yanmeng Shi (ORCID: https://orcid.org/0009-0005-5933-7753)
- Jonathan Sussman (ORCID: https://orcid.org/0000-0002-3057-3550)
- Xiaohua Wu
- Mirit I. Aladjem
- Chia-Yu Chang
- Hailong Wang
- Te-Hsuan Bu
Institutions
- Scripps Research Institute (US)
- Tianjin Normal University (CN)
- San Diego Biomedical Research Institute (US)
- National Cancer Institute (US)
- Center for Cancer Research (US)
- Capital Normal University (CN)
Publication Details
- Journal
- bioRxiv (Cold Spring Harbor Laboratory)
- Published
- 2026-01-16
- DOI
- https://doi.org/10.64898/2026.01.15.699632
- Primary Topic
- DNA Repair Mechanisms
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Massachusetts Institute of Technology
- Wake Forest University
- National Natural Science Foundation of China
- National Institutes of Health
- University of North Carolina at Chapel Hill
- University of California, San Francisco
- National Cancer Institute