Polλ 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 (Pol theta) and occur independently of nonhomologous end-joining (NHEJ) factors. An unresolved question is whether MMEJ is facilitated by a single Pol theta-mediated end-joining pathway or consists of additional undiscovered pathways. We find that human X-family Pol lambda, which functions in NHEJ, additionally exhibits robust MMEJ activity like Pol theta. Pol lambda promotes MMEJ in mammalian cells independently of essential NHEJ factors LIG4/XRCC4 and Pol theta, which reveals a distinct Pol lambda-dependent MMEJ mechanism. X-ray crystallography employing in situ photo-induced DSB formation captured Pol lambda in the act of stabilizing a microhomology-mediated DNA synapse with incoming nucleotide at 2.0 & ANGS; resolution and reveals how Pol lambda performs replication across a DNA synapse joined by minimal base-pairing. Last, we find that Pol lambda is semisynthetic lethal with BRCA1 and BRCA2. Together, these studies indicate Pol lambda MMEJ as a distinct DSB repair mechanism.