| Chromosomal translocations in hematologic and mesenchymal tumors form overwhelmingly by nonhomologous end-joining (NHEJ). Canonical NHEJ, essential for the repair of radiation-induced and some programmed double-strand breaks (DSBs), requires the Xrcc4--ligase IV complex. For other DSBs, the requirement for Xrcc4--ligase IV is less stringent, suggesting the existence of alternative end-joining (alt-NHEJ) pathways. To understand the contributions of the canonical NHEJ and alt-NHEJ pathways, we examined translocation formation in cells deficient in Xrcc4--ligase IV. We found that Xrcc4--ligase IV is not required for but rather suppresses translocations In addition to Xrcc4--ligase IV, mammalian cells have 2 more ATP-dependent DNA ligases, DNA ligase I and DNA ligase III, which are required for DNA replication and repair. Ligase III is also implicated in alt-NHEJ of DSBs in plasmids. Deletion of ligase III results in early embryonic lethality in the mouse, as well as apparent cellular lethality, which has precluded definitive characterization of ligase III function. We used pre-emptive complementation to determine the viability requirement for ligase III in mammalian cells and its requirement in DNA repair. Various forms of ligase III were introduced stably into mouse embryonic stem (ES) cells containing a conditional allele of ligase III that could be deleted with Cre recombinase With this approach, we find that the mitochondrial, but not nuclear, ligase III is required for cellular viability. Although the catalytic function of ligase III is required, the zinc finger (ZnF) and BRCT domains of ligase III are not. The viability requirement for ligase III can be circumvented by targeting ligase Ito the mitochondria or expressing DNA ligase from Chlorella virus, the minimal eukaryal nick-sealing enzyme, or from Escherichia coli, an NAD+-dependent ligase. Ligase III null cells are not sensitive to several DNA damaging agents that sensitize XRCC1-deficient cells. In order to understand whether alt-NHEJ is impaired in ligase III null cells, we induced chromosomal translocations by utilizing zinc finger endonucleases. We found that in the absence of ligase III, translocations are reduced in frequency and translocation breakpoint junctions show less microhomology. Our results establish a role for ligase III in mitochondria and in alt-NHEJ, but distinguish it from its interacting protein XRCC1. |
