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Crucial role for DNA ligase III in mitochondria but not in Xrcc1-dependent repair

Abstract

Mammalian cells have three ATP-dependent DNA ligases, which are required for DNA replication and repair1. 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 lethality2, 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 (mES) 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 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 enzyme3, or Escherichia coli LigA, an NAD+-dependent ligase1. Lig3-null cells are not sensitive to several DNA-damaging agents that sensitize Xrcc1-deficient cells4,5,6. Our results establish a role for Lig3 in mitochondria, but distinguish it from its interacting protein Xrcc1.

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Figure 1: Mitochondrial Lig3 activity is critical for cellular viability.
Figure 2: Mitochondrial DNA ligase activity can be provided by a variety of DNA ligases.
Figure 3: Loss of Lig3 is not associated with sensitivity to several DNA-damaging agents or with increased SCE.

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Acknowledgements

We thank K. Caldecott for the gift of the Lig3 expression vector, and M. Sanz for initial assistance with the SCE analysis. We also thank the members of Jasin laboratory, especially Y. Akamatsu, J. LaRocque, E. Kass and F. Vanoli, for discussions. This work was supported by PA CURE (to B.V.H.) and by National Institutes of Health grants ES019566 (to B.V.H.), NS37956 and CA21765 (to P.J.M.), and GM54668 (to M.J.).

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Contributions

D.S. performed most of the experiments. D.S. and M.J. designed the research and wrote the paper. A.F. performed the long-range qPCR assays to investigate mitochondrial BER and mitochondrial DNA maintenance, and with B.V.H. analysed the data. Y.G. and P.J.M. designed the initial Lig3 targeting scheme and generated the Lig3wt/cKO embryonic stem cells. J.A. and A.-K.H. acquired confocal images for GFP-tagged proteins. E.B. contributed technical assistance and preparation of the manuscript. S.S. contributed discussions, provided reagents and shared unpublished data.

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Correspondence to Maria Jasin.

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The authors declare no competing financial interests.

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Simsek, D., Furda, A., Gao, Y. et al. Crucial role for DNA ligase III in mitochondria but not in Xrcc1-dependent repair. Nature 471, 245–248 (2011). https://doi.org/10.1038/nature09794

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