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Mutagenesis. 2017 Mar 1;32(2):245-256. doi: 10.1093/mutage/gew042.

Mycobacterium tuberculosis and Mycobacterium marinum non-homologous end-joining proteins can function together to join DNA ends in Escherichia coli.

Author information

1
Department of Molecular and Cellular Physiology.
2
Present address: Jefferson College of Health Sciences, 101 Elm Avenue, S.E., Roanoke, VA 24013, USA.
3
Department of Medicine and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71103, USA.
4
Department of Biology, University of Louisiana at Lafayette, PO Box 42451, Lafayette, LA 70504, USA.
5
Present address: Joan C. Edwards School of Medicine, Marshall University, 1600 Medical Center Drive, Huntington, WV 25701, USA.

Abstract

Mycobacterium tuberculosis and Mycobacterium smegmatis express a Ku protein and a DNA ligase D and are able to repair DNA double strand breaks (DSBs) by non-homologous end-joining (NHEJ). This pathway protects against DNA damage when bacteria are in stationary phase. Mycobacterium marinum is a member of this mycobacterium family and like M. tuberculosis is pathogenic. M. marinum lives in water, forms biofilms and infects fish and frogs. M. marinum is a biosafety level 2 (BSL2) organism as it can infect humans, although infections are limited to the skin. M. marinum is accepted as a model to study mycobacterial pathogenesis, as M. marinum and M. tuberculosis are genetically closely related and have similar mechanisms of survival and persistence inside macrophage. The aim of this study was to determine whether M. marinum could be used as a model to understand M. tuberculosis NHEJ repair. We identified and cloned the M. marinum genes encoding NHEJ proteins and generated E. coli strains that express the M. marinum Ku (Mm-Ku) and ligase D (Mm-Lig) individually or together (LHmKumLig strain) from expression vectors integrated at phage attachment sites in the genome. We demonstrated that Mm-Ku and Mm-Lig are both required to re-circularize Cla I-linearized plasmid DNA in E. coli. We compared repair of strain LHmKumLig with that of an E. coli strain (BWKuLig#2) expressing the M. tuberculosis Ku (Mt-Ku) and ligase D (Mt-Lig), and found that LHmKumLig performed 3.5 times more repair and repair was more accurate than BWKuLig#2. By expressing the Mm-Ku with the Mt-Lig, or the Mt-Ku with the Mm-Lig in E. coli, we have shown that the NHEJ proteins from M. marinum and M. tuberculosis can function together to join DNA DSBs. NHEJ repair is therefore conserved between the two species. Consequently, M. marinum is a good model to study NHEJ repair during mycobacterial pathogenesis.

PMID:
27613236
PMCID:
PMC5989629
DOI:
10.1093/mutage/gew042
[Indexed for MEDLINE]
Free PMC Article

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