Structural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining

Nonhomologous end joining (NHEJ) is the primary pathway of vertebrate DNA double-strand-break repair. NHEJ polymerases and nucleases can modify DNA ends to render them compatible for ligation, but these enzymes are usually deployed only when necessary for repair of damaged DNA ends, thereby minimizing mutagenesis. Using frog egg extracts, we reveal a structural role for the NHEJ-specific DNA Ligase IV (Lig4) in promoting NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the short-range synaptic complex, in which DNA ends are closely aligned prior to ligation. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of short-range synapsis. In this way, compatible ends can be rapidly ligated without polymerase or nuclease activity, which we previously showed is restricted to the short-range synaptic complex. Our results provide a molecular basis for the fidelity of NHEJ.