Beyond Short Microhomologies: Mismatch-Compatible Pol θ-Mediated DNA Damage Repair

DNA polymerase θ (Pol θ)-mediated end-joining (TMEJ), one of several pathways for repairing DNA double-strand breaks, is traditionally thought to initiate via anchoring at short, consecutive, and perfectly matched microhomologies (MHs). Emerging evidence indicates that Pol θ can utilize MHs containing mismatches both in vitro and in vivo. This revised definition of MH provides a mechanistic explanation for a broader spectrum of Pol θ-dependent repair outcomes. Here, we summarize recent findings on the revised definition of MHs utilized by Pol θ, assess the applicability of this concept across species, and compare TMEJ with other (micro)hom(e)ology-mediated repair pathways. We explore how mismatch-containing MHs expand Pol θ-associated mutational signatures and provide a framework for future studies on Pol θ’s role in DNA repair and cancer biology.