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Mechanism of nonhomologous end-joining in mycobacteria: a low-fidelity repair system driven by Ku, ligase D and ligase C

Nature Structural & Molecular Biology volume 12, pages 304–312 (2005)Cite this article

Abstract

DNA double-strand breaks (DSBs) can be repaired either via homologous recombination (HR) or nonhomologous end-joining (NHEJ). Both pathways are operative in eukaryotes, but bacteria had been thought to rely on HR alone. Here we provide direct evidence that mycobacteria have a robust NHEJ pathway that requires Ku and a specialized polyfunctional ATP-dependent DNA ligase (LigD). NHEJ of blunt-end and complementary 5′-overhang DSBs is highly mutagenic (∼50% error rate). Analysis of the recombination junctions ensuing from individual NHEJ events highlighted the participation of several DNA end-remodeling activities, including template-dependent fill-in of 5′ overhangs, nontemplated addition of single nucleotides at blunt ends, and nucleolytic resection. LigD itself has the template-dependent and template-independent polymerase functions in vitro that compose the molecular signatures of NHEJ in vivo. Another ATP-dependent DNA ligase (LigC) provides a backup mechanism for LigD-independent error-prone repair of blunt-end DSBs. We speculate that NHEJ allows mycobacteria to evade genotoxic host defense.

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Figure 1: Multiple DNA ligases of Mycobacterium smegmatis.
Figure 2: NHEJ of blunt-end DSBs in M. smegmatis depends on LigD and is error-prone.
Figure 3: Molecular outcome of imprecise NHEJ events at blunt DSBs.
Figure 4: NHEJ of 5′ cohesive ends is imprecise and depends on LigD.
Figure 5: Outcome of imprecise NHEJ events at 5′-overhang DSBs.
Figure 6: Ligase and polymerase activities of recombinant mycobacterium LigD.
Figure 7: Mycobacterial NHEJ requires Ku.

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Acknowledgements

This research was supported by US National Institutes of Health grants AI53417 (to M.S.G.) and GM63611 (to S.S.). M.S.G. is the recipient of research awards from the Ellison Medical Foundation and the New York Academy of Medicine Speakers Fund for Biomedical Research. S.S. is an American Cancer Society Research Professor.

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Author notes

  1. Chunling Gong and Paola Bongiorno: These authors contributed equally to this work.

Authors and Affiliations

  1. Immunology and Molecular Biology Programs, Sloan-Kettering Institute, New York, 10021, New York, USA

    Chunling Gong, Paola Bongiorno, Alexandra Martins, Nicolas C Stephanou, Hui Zhu, Stewart Shuman & Michael S Glickman

  2. Division of Infectious Diseases, Memorial Sloan Kettering Cancer Center, New York, 10021, New York, USA

    Chunling Gong, Paola Bongiorno, Alexandra Martins, Nicolas C Stephanou, Hui Zhu, Stewart Shuman & Michael S Glickman

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Correspondence to Stewart Shuman or Michael S Glickman.

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Supplementary information

Supplementary Table 1

Mycobacterium smegmatis deletion strains. (PDF 19 kb)

Supplementary Methods (PDF 89 kb)

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Gong, C., Bongiorno, P., Martins, A. et al. Mechanism of nonhomologous end-joining in mycobacteria: a low-fidelity repair system driven by Ku, ligase D and ligase C. Nat Struct Mol Biol 12, 304–312 (2005). https://doi.org/10.1038/nsmb915

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