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Microhomology Mediated End JoiningEdit

Microhomology mediated end joining (MMEJ) is a DNA repair process that provides a backup route for fixing double-strand breaks (DSBs) when the primary systems are unavailable or overwhelmed. This pathway relies on short regions of microhomology—tiny stretches of matching bases—to align broken DNA ends before they are sealed. Because the alignment depends on these brief homologies and because the repair process often trims or deletes sequences flanking the break, MMEJ is considered more error-prone than other major DSB repair pathways. In many cells, MMEJ acts as a supplementary mechanism that helps preserve genome integrity under stress, but its activity can also contribute to genomic instability and mutagenesis when left unchecked. For readers, it is important to see MMEJ as part of a broader repair landscape that includes more precise options, but becomes favored under certain cellular conditions.

MMEJ sits alongside classical non-homologous end joining (non-homologous end joining) and homologous recombination (homologous recombination) as part of the cell’s toolkit for DSB repair. While NHEJ quickly ligates ends with minimal processing, and HR uses a homologous template to restore sequence faithfully, MMEJ involves resection to expose microhomologies, anneals these microhomologous regions, trims overhangs, and fills in gaps before ligation. The end result is often a junction accompanied by deletions of the sequences between the break and the microhomology, making MMEJ a source of structural variation in genomes. In this way, MMEJ can be seen as a distinct repair modality with characteristic fingerprints in sequencing data, but it frequently overlaps with what some researchers refer to as alternative end joining (A-EJ), a term used to describe error-prone backup pathways that come into play when the canonical routes are compromised.

Mechanism

  • End processing and resection: DSB ends undergo limited resection to reveal short stretches of microhomology. Key players in this step include resection factors such as MRE11 and CtIP, which prepare DNA ends for subsequent steps. MRE11 and CtIP help generate the single-stranded overhangs that expose microhomologous sequences.

  • Microhomology search and annealing: The exposed microhomologies (often 2–25 base pairs) align the broken ends. This annealing step is a defining feature of MMEJ and sets it apart from other repair modes that rely on blunt-end ligation or longer homologous templates.

  • Flap trimming and gap filling: When microhomologies anneal, unpaired flaps and overhangs require trimming. Enzymes such as FEN1 contribute to removing these flaps, while a specialized DNA polymerase fills in the remaining gaps.

  • Ligation: The final seam is sealed by a ligase complex, most commonly involving DNA ligase III in partnership with XRCC1. This ligase choice helps explain the propensity of MMEJ repairs to leave behind deletions and to generate junctions distinct from those produced by NHEJ or HR. See how this pathway interplays with other ligases and scaffolding factors in the broader network of DNA repair.

  • Key polymerase: A central component of MMEJ is DNA polymerase theta (POLQ). POLQ carries both a polymerase activity that extends DNA to bridge gaps and, in many organisms, a helicase-like function that facilitates strand annealing and end-joining. In cells where POLQ is absent or inhibited, MMEJ efficiency drops, underscoring POLQ’s critical role in this pathway.

Key players

  • POLQ (DNA polymerase theta): The enzyme most closely associated with MMEJ activity in many eukaryotic cells; its activity is tightly linked to microhomology-based repair and to the mutagenic outcomes of MMEJ.

  • LIG3-XRCC1 complex: The ligase and its adaptor that typically completes the final nick sealing step in MMEJ.

  • FEN1: A structure-specific nuclease involved in removing flaps created during microhomology annealing and processing the repair junction.

  • MRE11, CtIP: Initiators and organizers of end resection that uncover microhomologies and set the stage for annealing.

  • RAD52: A factor that can assist annealing in some contexts, particularly in systems where RAD52’s strand-annealing activity supports microhomology-based pairing.

  • Other factors: In certain cells or organisms, additional proteins that influence resection, annealing efficiency, or ligation can modulate MMEJ activity, illustrating that the pathway is embedded within a broader repair network.

Relationship to other pathways

  • Comparison with NHEJ: NHEJ is typically rapid and relatively gene-friendly, joining ends with minimal processing. MMEJ, by contrast, depends on exposed microhomologies and often introduces deletions, making it a more error-prone option that is invoked when NHEJ is unavailable or insufficient.

  • Comparison with HR: HR uses a homologous sequence as a template for error-free repair. MMEJ is more limited in its accuracy and usually results in sequence loss around the break, contrasting with the high-fidelity restoration accomplished by HR.

  • Alternative end joining (A-EJ): Some researchers use A-EJ to refer to a family of error-prone end-joining processes, including MMEJ as a prominent example. The exact boundaries between MMEJ and other A-EJ pathways are a topic of ongoing investigation, with debates about how to classify and distinguish these mechanisms across species and cellular states.

  • Role in replication stress and cancer: In cells experiencing replication stress or in tumors with HR defects (for example, BRCA1/2 deficiencies), MMEJ can become more prominent. This shift has implications for genome stability and for therapeutic strategies that target POLQ or related components.

Biological and clinical relevance

  • Genome stability and mutagenesis: Because MMEJ often deletes sequence between the break and the microhomology, it can contribute to structural variation, copy number changes, and chromosomal rearrangements. These outcomes are detectable in cancer genomes and in other settings of genomic instability.

  • Cancer biology: Elevated POLQ expression and MMEJ activity have been observed in certain cancers, and this association can correlate with prognosis and treatment response. The reliance of HR-deficient tumors on MMEJ creates a potential vulnerability exploitable by targeted therapies.

  • Therapeutic implications: Inhibitors targeting POLQ are under investigation as cancer therapies, aiming to create synthetic lethality in tumors with compromised HR. By inhibiting POLQ, researchers hope to limit the tumor cell’s compensatory repair options, increasing sensitivity to DNA-damaging agents or radiotherapy. Early studies in cell culture and animal models inform ongoing drug development strategies, including combinations with PARP inhibitors and other DNA damage response modulators.

  • Genome editing considerations: In modern genome engineering, understanding MMEJ helps interpret outcomes from nuclease-induced DSBs (for example, those produced by CRISPR systems). Since MMEJ tends to yield deletions at microhomology regions, its activity can shape the spectrum of edits and guide design decisions for desired outcomes.

Controversies and debates

  • Distinct pathway vs. backup mode: A central discussion in the field concerns whether MMEJ should be treated as a standalone repair pathway with its own dedicated machinery, or primarily as a backup mode that becomes apparent when NHEJ and HR are compromised. The line between MMEJ and broader A-EJ concepts is not always sharp, and different laboratories emphasize different defining features (end resection, microhomology length, ligation dependencies).

  • Quantifying MMEJ versus other end-joining: Measuring the relative contributions of MMEJ in cells and tissues remains technically challenging. Assays based on reporter constructs or sequencing junctions can yield varying estimates depending on cell type, phase of the cell cycle, and the presence of repair inhibitors or mutations in key factors.

  • Species-specific variation: The extent to which MMEJ operates as a major repair pathway can differ across organisms. While POLQ is widely implicated in MMEJ in many eukaryotes, the exact set of required factors and the balance with other end-joining routes can vary, raising questions about how universal the canonical MMEJ model is.

  • Therapeutic targeting and resistance: While POLQ inhibitors hold promise for exploiting synthetic lethality in HR-deficient cancers, tumors may adapt by upregulating alternative repair pathways or by mutating other components of the repair network. The long-term efficacy and potential resistance mechanisms are active topics of research.

Evolution and diversity

MMEJ-like mechanisms appear across a wide range of eukaryotic species, reflecting evolutionary pressure to preserve genome integrity under stress while accommodating repair flexibility. The reliance on POLQ and microhomology is a recurring theme, but organism-specific differences in accessory factors shape how prominently MMEJ features in the overall repair landscape. This diversity helps explain why experimental observations about MMEJ can vary between model organisms and human cells.

See also