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Journal of Bacteriology, July 2007, p. 5237-5246, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.00332-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mycobacterial Nonhomologous End Joining Mediates Mutagenic Repair of Chromosomal Double-Strand DNA Breaks{triangledown}

Nicolas C. Stephanou,1,4 Feng Gao,1 Paola Bongiorno,1 Sabine Ehrt,4 Dirk Schnappinger,5 Stewart Shuman,2,4,5 and Michael S. Glickman1,3,4*

Immunology Program,1 Molecular Biology Program,2 Division of Infectious Diseases, Memorial Sloan-Kettering Cancer Center, New York, New York 10021,3 Program in Immunology and Microbial Pathogenesis, Weill Graduate School of Medical Sciences of Cornell University, New York, New York,4 Program in Molecular Biology, Weill Graduate School of Medical Sciences of Cornell University, New York, New York5

Received 5 March 2007/ Accepted 30 April 2007

Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient Mycobacterium smegmatis strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An M. smegmatis {Delta}recA {Delta}ku double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs.

* Corresponding author. Mailing address: Box 9, Division of Infectious Diseases, Memorial Sloan-Kettering Cancer Center, New York, NY 10021. Phone: (646) 888-2368. Fax: (646) 422-0516. E-mail: glickmam{at}mskcc.org

{triangledown} Published ahead of print on 11 May 2007.

Journal of Bacteriology, July 2007, p. 5237-5246, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.00332-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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