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Mammalian cells have three ATP-dependent DNA ligases, which are required for DNA replication and repair (1). Homologues 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 (ref. 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 preemptive 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 (mES) cells containing a conditional allele of Lig3that 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 BRCA1 carboxy (C)-terminal-related (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.sup.+]-dependent ligase (1). Lig3-null cells are not sensitive to several DNA-damaging agents that sensitize Xrccl-deficient cells (4-6). Our results establish a role for Lig3 in mitochondria, but distinguish it from its interacting protein Xrcc1., Biochemical and cell biological experiments implicate the nuclear Lig3-Xrcc1 complex in single-strand break repair, short patch base excision repair and nucleotide excision repair (1). Lig3 and Xrcc1 interact through carboxy [...]

DNA replication and repair in mammalian cells involves three distinct DNA ligases: ligase I (Ligl), ligase III (Lig3) and ligase IV (Lig4) (1). Lig3 is considered a key ligase during base excision repair because its stability depends upon its nuclear binding partner Xrcc1, a critical factor for this DNA repair pathway (2,3). Lig3 is also present in the mitochondria, where its role in mitochondrial DNA (mtDNA) maintenance is independent of Xrcc1 (ref. 4). However, the biological role of Lig3 is unclear as inactivation of murine Lig3 results in early embryonic lethality (5). Here we report that Lig3 is essential for mtDNA integrity but dispensable for nuclear DNA repair. Inactivation of Lig3 in the mouse nervous system resulted in mtDNA loss leading to profound mitochondrial dysfunction, disruption of cellular homeostasis and incapacitating ataxia. Similarly, inactivation of Lig3 in cardiac muscle resulted in mitochondrial dysfunction and defective heart-pump function leading to heart failure. However, Lig3 inactivation did not result in nuclear DNA repair deficiency, indicating essential DNA repair functions of Xrcc1 can occur in the absence of Lig3. Instead, we found that Lig1 was critical for DNA repair, but acted in a cooperative manner with Lig3. Additionally, Lig3 deficiency did not recapitulate the hallmark features of neural Xrcc1 inactivation such as DNA damage-induced cerebellar interneuron loss (6), further underscoring functional separation of these DNA repair factors. Therefore, our data reveal that the critical biological role of Lig3 is to maintain mtDNA integrity and not Xrcc1-dependent DNA repair., Lig3 has been ascribed repair functions during DNA replication, DNA strand break repair through the BER/single-strand break repair pathway, and has been implicated in double-strand break repair if non-homologous end-joining [...]

DNA double-strand breaks (DSBs) are generated by the recombination activating gene (RAG) endonuclease in all developing lymphocytes as they assemble antigen receptor genes (1). DNA cleavage by RAG occurs only at the G1 phase of the cell cycle and generates two hairpin-sealed DNA (coding) ends that require nucleolytic opening before their repair by classical non-homologous end-joining (NHEJ) (1-3). Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently (2,3). Here, in vivo, we show that in murine cells the histone protein H2AX prevents nucleases other than Artemis from processing hairpin-sealed coding ends; in the absence of H2AX, CtIP can efficiently promote the hairpin opening and resection of DNA ends generated by RAG cleavage. This CtIP-mediated resection is inhibited by γ-H2AX and by MDC-1 (mediator of DNA damage checkpoint 1), which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ataxia telangiectasia mutated (ATM) kinase activates antagonistic pathways that modulate this resection. CtIP DNA end resection activity is normally limited to cells at post-replicative stages of the cell cycle, in which it is essential for homology-mediated repair (4,5). In G1-phase lymphocytes, DNA ends that are processed by CtIP are not efficiently joined by classical NHEJ and the joints that do form frequently use micro-homologies and show significant chromosomal deletions. Thus, H2AX preserves the structural integrity of broken DNA ends in G1-phase lymphocytes, thereby preventing these DNA ends from accessing repair pathways that promote genomic instability., V(D)J recombination, the reaction that assembles the second exon of all antigen receptor genes, requires the generation and repair of DNA DSBs and occurs exclusively during the G1 phase of [...]