biostudies-literatureChandramouly GNCI NIH HHSNIGMS NIH HHS107-114https://www.ebi.ac.uk/biostudies/studies/S-EPMC10197178biostudies-literatureUnknown30(1)The double-strand break (DSB) repair pathway called microhomology-mediated end-joining (MMEJ) is thought to be dependent on DNA polymerase theta (Polθ) and occur independently of nonhomologous end-joining (NHEJ) factors. An unresolved question is whether MMEJ is facilitated by a single Polθ-mediated end-joining pathway or consists of additional undiscovered pathways. We find that human X-family Polλ, which functions in NHEJ, additionally exhibits robust MMEJ activity like Polθ. Polλ promotes MMEJ in mammalian cells independently of essential NHEJ factors LIG4/XRCC4 and Polθ, which reveals a distinct Polλ-dependent MMEJ mechanism. X-ray crystallography employing in situ photo-induced DSB formation captured Polλ in the act of stabilizing a microhomology-mediated DNA synapse with incoming nucNature structural & molecular biologyPolλ promotes microhomology-mediated end-joining.PMC10197178R01 GM130889P30 CA006927UH2 CA271230R01 GM137124Pomerantz RTCalbert MLTredinnick TDemidova EVOzdemir AYTyagi MJamsen JWilson SHKent TBorisonnik NChandramouly GArora SfalsePolλ promotes microhomology-mediated end-joining.The double-strand break (DSB) repair pathway called microhomology-mediated end-joining (MMEJ) is thought to be dependent on DNA polymerase theta (Polθ) and occur independently of nonhomologous end-joining (NHEJ) factors. An unresolved question is whether MMEJ is facilitated by a single Polθ-mediated end-joining pathway or consists of additional undiscovered pathways. We find that human X-family Polλ, which functions in NHEJ, additionally exhibits robust MMEJ activity like Polθ. Polλ promotes MMEJ in mammalian cells independently of essential NHEJ factors LIG4/XRCC4 and Polθ, which reveals a distinct Polλ-dependent MMEJ mechanism. X-ray crystallography employing in situ photo-induced DSB formation captured Polλ in the act of stabilizing a microhomology-mediated DNA synapse with incoming nuc2023-01-01T00:00:00Z2023 Jan2025-04-04T10:19:38.885Z2025-02-19T00:12:15.933ZS-EPMC101971783653610410.1038/s41594-022-00895-4