Crucial roles for DNA ligase III in mitochondria but not in XRCC1-dependent repair

Mammalian cells have 3 ATP-dependent DNA ligases, which are required for DNA replication and repair(1). Homologs of ligase I (Lig1) and ligase IV (Lig4) are ubiquitous in eukarya, whereas ligase III (Lig3), which has nuclear and mitochondrial forms, appears to be restricted to vertebrates. Lig3 is implicated in various DNA repair pathways with its partner protein XRCC1(1). Deletion of Lig3 results in early embryonic lethality in mice, as well as apparent cellular lethality(2), which has precluded definitive characterization of Lig3 function. Here we used pre-emptive complementation to determine the viability requirement for Lig3 in mammalian cells and its requirement in DNA repair. Various forms of Lig3 were introduced stably into mouse embryonic stem (ES) cells containing a conditional allele of Lig3 that could be deleted with Cre recombinase. With this approach, we find that the mitochondrial, but not nuclear, Lig3 is required for cellular viability. Although the catalytic function of Lig3 is required, the zinc finger (ZnF) and BRCT domains of Lig3 are not. Remarkably, the viability requirement for Lig3 can be circumvented by targeting Lig1 to the mitochondria or expressing Chlorella virus DNA ligase, the minimal eukaryal nick-sealing enzyme(3), or Escherichia coli LigA, an NAD+-dependent ligase(1). Lig3 null cells are not sensitive to several DNA damaging agents that sensitize XRCC1-deficient cells(4,5,6). Our results establish a role for Lig3 in mitochondria, but distinguish it from its interacting protein XRCC1.