Mycobacteria exploit three genetically distinct DNA double‐strand break repair pathways

Abstract

Bacterial pathogens rely on their DNA repair pathways to resist genomic damage inflicted by the host. DNA double-strand breaks (DSBs) are especially threatening to bacterial viability. DSB repair by homologous recombination (HR) requires nucleases that resect DSB ends and a strand exchange protein that facilitates homology search. RecBCD and RecA perform these functions in E. coli and constitute the major pathway of error free DSB repair. Mycobacteria, including the human pathogen M. tuberculosis, elaborate an additional error-prone pathway of DSB repair via nonhomologous end-joining (NHEJ) catalyzed by Ku and DNA ligase D (LigD). Little is known about the relative contributions of HR and NHEJ to mycobacterial chromosome repair, the factors that dictate pathway choice, or the existence of additional DSB repair pathways. Here we demonstrate that Mycobacterium smegmatis has three DSB repair pathway options: HR, NHEJ, and a novel mechanism of single-strand annealing (SSA). Inactivation of NHEJ or SSA is compensated by elevated HR. We find that mycobacterial RecBCD does not participate in HR or confer resistance to ionizing radiation (IR), but is required for the RecA-independent SSA pathway. In contrast, the mycobacterial helicase-nuclease AdnAB participates in the RecA-dependent HR pathway, and is a major determinant of resistance to IR and oxidative DNA damage. These findings reveal distinctive features of mycobacterial DSB repair, most notably the dedication of the RecBCD and AdnAB helicase-nuclease machines to distinct repair pathways.

DNA Ligase C1 Mediates the LigD-Independent Nonhomologous End-Joining Pathway of Mycobacterium smegmatis

“…However, additional pathways of DSB repair operate in other bacteria, including mycobacteria, such as Mycobacterium smegmatis and M. tuberculosis. M. smegmatis elaborates three genetically distinct DNA repair pathways: RecA-dependent HR, single-strand annealing (SSA), and nonhomologous end joining (NHEJ) (6). All mycobacterial HR uses the RecA strand exchange protein, whereas two subpathways utilize the AdnAB helicase-nuclease (6,7) or RecO (8), respectively.…”

“…M. smegmatis elaborates three genetically distinct DNA repair pathways: RecA-dependent HR, single-strand annealing (SSA), and nonhomologous end joining (NHEJ) (6). All mycobacterial HR uses the RecA strand exchange protein, whereas two subpathways utilize the AdnAB helicase-nuclease (6,7) or RecO (8), respectively. The SSA pathway requires RecBCD (6), which does not participate in HR in mycobacteria, and RecO (8).…”

“…Mycobacterial NHEJ can recircularize transformed linear plasmids (9)(10)(11)(12)(13)(14), repair IR-induced DSBs in late-stationary-phase cells (15,16), protect mycobacteria from desiccation (15), and seal homing endonuclease-induced DSBs (6,16). In all of these experimental systems, the NHEJ pathway requires the DNA end binding protein Ku and the polyfunctional DNA ligase LigD.…”

“…Similarly, M. smegmatis ⌬Ku or ⌬ligD bacteria in late stationary phase but not log phase are sensitized to IR (16) and cannot repair I-SceI-induced chromosomal breaks by NHEJ (6). Ablation of ku or ligD also leads to reciprocal upregulation of the HR pathway in the I-SceI system, suggesting that the NHEJ and HR pathways compete for repair of DSBs in log-phase cells (6).…”

The DNA Repair Repertoire of Mycobacterium smegmatis FenA Includes the Incision of DNA 5′ Flaps and the Removal of 5′ Adenylylated Products of Aborted Nick Ligation

“…Rather, the flap endonuclease and AppDNA removal activities of FenA point toward a role in gap repair during lagging-strand DNA replication or NHEJ. The flap removal activity of FenA might also come into play in the single-strand annealing (SSA) pathway of DNA double-strand break (DSB) repair that entails large DNA deletions between homologous regions flanking the DSB (31).…”