Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break

Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase micro (Polmicro) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polmicro engaging a DSB substrate. Synapsis is mediated solely by Polmicro, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol micro complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polmicro may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.