Mycobacterial nonhomologous end joining mediates mutagenic repair of chromosomal double-strand DNA breaks

Abstract

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.

FIG. 1.

UV resistance of NHEJ- and HR-deficient strains. Wild-type, NHEJ-deficient (Δku, ΔligD, or ΔligD ku), HR-deficient (ΔrecA or ΔrecBCD), or combined HR/NHEJ-deficient strains (ΔrecBCD ΔligD or ΔrecA Δku) were exposed to high-dose (A) and low-dose (B) UV irradiation, and survival was plotted on a log scale y axis as a percentage compared to unexposed cells of the same strain. Error bars ± standard errors of the means. WT, wild type.

FIG. 2.

NHEJ is required for resistance to IR in late stationary phase. Wild-type, NHEJ-deficient, HR-deficient, or HR/NHEJ-deficient strains were exposed to gamma irradiation from a ¹³⁷Cs source, and survival was calculated by enumerating surviving colonies compared to a mock-exposed control sample of the same strain. Panel A depicts cells harvested from logarithmically growing cultures in LB medium. Panel B compares logarithmic- and early-stationary-phase cells of wild-type, ΔrecA, and ΔrecA Δku strains in LB medium. Panel C shows late-stationary-phase cells grown in LB medium. Panel D shows late-stationary-phase cells grown in 7H9 medium. For panels C and D, the symbols are identical to those in panel A. Error bars are ± standard errors of the means. WT, wild type.

FIG. 3.

MMS sensitivity of NHEJ- and HR-deficient strains. Serial dilutions of the indicated strains were cultured on agar medium containing an MMS dose ranging from 0.001 to 0.1%, and survival was calculated compared to unexposed control strains. Error bars are ± standard errors of the means. WT, wild type.

FIG. 4.

Conditionally expressed I-SceI efficiently cleaves the M. smegmatis chromosome at a supplied I-SceI site. (A) Plasmid map of pMSG375 which contains the I-SceI homing endonuclease with an N-terminal HA epitope tag under the control of Pmyc1 TetO, which confers tetracycline-inducible expression when tetR is coexpressed. The plasmid also contains a single I-SceI site and an integrase which catalyzes site-specific integration at the attB site in the mycobacterial chromosome. When integrated at attB, all plasmid sequences are contained on the chromosome, thereby creating a single unique I-SceI cleavage site. (B) AHT-dependent expression of HA-I-SceI. The indicated strains transformed with the tetracycline-inducible HA-I-SceI construct were cultured with or without 50 ng/ml AHT. Whole-cell extracts were analyzed by Western blotting with an anti-HA monoclonal antibody. The predicted size of the HA-I-SceI protein is 29.4 kDa. The anti-DlaT antibody serves as a loading control. (C) Map of the I-SceI expression cassette when integrated at the attB locus with probe location and predicted sizes of bands by Southern blotting. (D) I-SceI cleaves the mycobacterial chromosome. Southern analysis of genomic DNA prepared from M. smegmatis transformed with pMSG375 without (−) and with (+) AHT for 0.5, 1, and 2 h after AHT addition. The probe spans the I-SceI site at attB, yielding a band of 800 bp with I-SceI cleavage (Cut). wt, wild type.

FIG. 5.

NHEJ is required for mutagenic repair and survival after I-SceI chromosomal DSBs. (A) The indicated wild-type or mutant M. smegmatis strains bearing pMSG375 were grown in the absence of tetracycline induction, and then serial dilutions were plated on 50 ng/ml AHT. Survival was calculated compared to control plates without AHT. Error bars are ± standard deviations. (B and C) Molecular outcomes of repair of I-SceI-induced chromosomal breaks in HR- and NHEJ-deficient strain backgrounds. (B) For each strain, the number of independent repair events analyzed (N) is noted in the first column. Numbers in each column are the number of wild-type or mutated sites, with percentages given in parentheses. For mutated sites, the number (percentages in parentheses) of deletions or additions is recorded, followed by the frequency of each addition event. The numbers (1 to 3) refer to the additions depicted in panel C. The top sequence in panel C is the wild-type I-SceI site. WT, wild type.

FIG. 6.

(A) Molecular characteristics of deletions in wild-type and recA-deficient strains. Nucleotides of the original I-SceI site are in blue, resected nucleotides are indicated by a line, and nucleotides of microhomology are boxed. To the right of the sequences is the number of events observed for each type of microhomology in wild-type and recA strains, with the number of deleted nucleotides in parentheses. (B) Statistical analysis of microhomology usage and deletion size in wild-type and recA-deficient strains. WT, wild type.