A groundbreaking study emerging from Scripps Research has unveiled an overlooked, yet critical mechanism that cancer cells exploit to survive the pervasive DNA damage encountered during replication. The enzyme DNA polymerase theta (Polθ), already a prominent target in cancer therapeutics, has now been recognized as a pivotal player in an alternative repair pathway directly acting at broken replication forks—one of the most recurrent forms of DNA injury in proliferating tumor cells. This discovery challenges prior assumptions by demonstrating that Polθ-driven microhomology-mediated end joining (MMEJ) actively repairs single-ended double-strand breaks at replication forks, rather than break-induced replication (BIR) being the exclusive frontline responder as formerly believed.
Replication forks are dynamic DNA structures arising during genome duplication where the DNA double helix unwinds, allowing enzymatic machinery to faithfully copy genetic information. However, when replication encounters obstacles such as endogenous lesions or replication stress, the replication fork can stall or collapse, resulting in single-ended double-strand breaks that pose a significant threat to genomic integrity. Traditionally, the cell was thought to predominantly employ BIR to mend such damage. BIR leverages an intact homologous DNA template to restart replication, thereby preserving genetic fidelity but operating at a relatively slow pace.
Contrasting with BIR, MMEJ is a more rapid, though error-prone, repair mechanism that aligns short homologous DNA sequences—microhomologies—to join broken DNA ends. Historically, MMEJ has been considered a backup mechanism, primarily mediating repair of double-ended breaks independent of replication. This new research overturns that paradigm by revealing that MMEJ, catalyzed by Polθ, is directly engaged at broken replication forks. Employing CRISPR nickase technology to precisely induce replication fork collapse and using sophisticated reporter systems alongside genome sequencing, the researchers identified unique mutational footprints inconsistent with canonical BIR but characteristic of fork-specific MMEJ.
These observations suggest that the MMEJ operating at replication forks—termed fork-MMEJ—differs mechanistically and functionally from its standard counterpart. Unlike canonical MMEJ, fork-MMEJ is initiated by the replication protein A (RPA), a single-stranded DNA-binding protein active during replication stress. This initiation results in asymmetric deletion patterns flanking the break site, creating a distinctive mutational signature frequently observed in various cancer genomes. Such error-prone repair may offer a double-edged sword: while compromising genome stability, it paradoxically confers tumors with resilience against lethal DNA damage, enabling continued proliferation under stress conditions.
Polθ’s central role in this process positions it as an even more compelling target for anticancer drug development than previously appreciated. Notably, tumors deficient in homologous recombination repair pathways, including those harboring BRCA1 or BRCA2 mutations, are especially reliant on Polθ-mediated MMEJ for survival. Inhibiting Polθ in these contexts disrupts the tumor cells’ ability to cope with replication-associated damage, selectively undermining their viability.
In a novel therapeutic insight, the study also highlights the interplay between fork-MMEJ and BIR regulated by the ATR kinase—a protein that senses DNA damage and orchestrates repair pathway choice. ATR suppresses fork-MMEJ, favoring the more accurate but slower BIR. Intriguingly, simultaneous inhibition of ATR and Polθ amplifies cancer cell death under replication stress conditions, with minimal adverse effects on normal cells. This synergy opens promising avenues for combination therapies harnessing existing ATR inhibitors alongside emerging Polθ inhibitors, potentially overcoming resistance mechanisms and improving clinical outcomes.
The profound implication of this research lies in reframing the timing and hierarchy of DNA repair mechanisms at replication forks. Whereas MMEJ was thought to function predominantly downstream or in backup capacities, it now appears integral to initial responses against replication-induced DNA breakage. This understanding could transform strategic drug targeting, emphasizing the disruption of Polθ activity at the earliest stages of DNA damage repair.
Looking forward, the Scripps Research team is expanding their investigation to elucidate additional protein components within the fork-MMEJ pathway. Each newly identified factor may represent a novel molecular target, broadening the landscape for therapeutic intervention. Moreover, they seek to deepen insights into ATR’s modulatory role, deciphering the molecular switches that balance repair pathway engagement and influence cell fate under replication stress.
This paradigm-shifting discovery not only advances fundamental knowledge of DNA damage responses in cancer biology but also redefines potential treatment strategies. By illuminating how cancer cells co-opt an error-prone yet expedient repair mechanism at replication forks, it opens doors to innovative therapies aimed at exploiting this vulnerability. The work underscores the criticality of targeting replication stress responses, a hallmark of cancer, to selectively eliminate tumor cells while sparing normal tissues.
As therapies evolve to incorporate precision targeting of DNA repair enzymes, understanding the nuanced differentiation between repair pathways becomes essential. Polθ’s newfound prominence reinforces the urgency of completing clinical development of inhibitors that can effectively disrupt the MMEJ process at replication forks. Combined with ATR inhibition approaches, such treatments promise a new frontier in cancer therapy, characterized by enhanced specificity and fewer side effects.
In summary, this compelling study marks a transformative advance in the field of cancer genomics and DNA repair. It challenges long-standing dogma, unveiling a previously underappreciated mechanism of fork-associated DNA repair mediated by Pol theta and MMEJ. This deeper mechanistic insight not only elucidates how tumors endure relentless replication stress but also provides a robust foundation for next-generation therapeutic strategies aimed at crippling cancer cells’ adaptive DNA repair capabilities.
Subject of Research: DNA repair mechanisms operative at broken replication forks in cancer cells, focusing on Pol theta–mediated microhomology-mediated end joining.
Article Title: Microhomology-mediated end joining acts directly on replication forks to repair single-ended double-strand breaks.
News Publication Date: 16-Mar-2026
Web References:
https://www.cell.com/molecular-cell/fulltext/S1097-2765(26)00130-9
http://dx.doi.org/10.1016/j.molcel.2026.02.016
References:
Li, S., Zhao, Y., Li, Y., Shah, S.B., Shi, Y., Nguyen, T., Bu, T-H., Loguercio, S., Sussman, J.H., Wang, Z., Chang, C-Y., Aladjem, M.I., Ray, A., Sasaki, T., Gilbert, D.M., Wang, H., Wu, X. (2026). Microhomology-mediated end joining acts directly on replication forks to repair single-ended double-strand breaks. Molecular Cell.
Image Credits: Scripps Research
Keywords: DNA damage response, replication fork collapse, Pol theta, microhomology-mediated end joining, break-induced replication, replication stress, DNA repair pathways, cancer therapeutics, ATR kinase, BRCA mutations, genome stability, replication-associated DNA breaks
Tags: alternative DNA repair pathways in cancerbreak-induced replication versus MMEJcancer cell DNA damage repaircancer therapeutics targeting PolθDNA polymerase theta functionDNA replication stress responsegenomic instability in tumor cellsmicrohomology-mediated end joining mechanismnovel cancer survival mechanismsreplication fork collapse repairreplication fork dynamics in cancersingle-ended double-strand break repair
