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

Abstract

Chromosomal DNA is under constant attack from clastogens that threaten the coding capacity and stability of the genome. Among the wide variety of DNA lesions that the cell must process, double strand breaks (DSBs) of DNA represent a particularly lethal form of DNA damage that must be repaired for chromosome replication and transcription to proceed. The sources of DSBs include ionizing radiation (IR), replication across a singlestranded nick (1), DNA processing enzymes that generate DSBs, such as topoisomerases, and embedded ribonucleotides in chromosomal DNA (2-5). As such, the repair of DSBs is a critical function in all living organisms, including bacteria. The most intensely studied and universally distributed pathway of DSB repair is RecA-dependent homologous recombination (HR), which uses an intact chromosomal template to repair the DSB without mutation. RecA-dependent HR is the dominant and sole DSB repair pathway in Escherichia coli. 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. The SSA pathway requires RecBCD (6), which does not participate in HR in mycobacteria, and RecO (8).Mycobacterial NHEJ can recircularize transformed linear plasmids (9-14), 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. Genetic ablation of ku or ligD reduces the efficiency of plasmid NHEJ by approximately 500-fold (10). 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 LigD protein has three autonomous enzymatic domains: polymerase (POL), phosphoesterase (PE), and ligase (LIG). LigD-POL is a primase-like polymerase that can add both templated and nontemplated deoxynucleoside triphosphates (dNTPs) or ribonucleoside triphosphates (rNTPs) to DNA substrates (13,17). Alanine substitution mutations at the diaspartate metal binding site of the polymerase (D136A/D138A) abolish polymerase activity in vitro (11). M. smegmatis expressing LigD-D136/138A cannot add nontemplated nucleotides to blunt end linear plasmid substrates, resulting in a rise in the fidelity of NHEJ with little change in the overall efficiency (10, 13). However, templated fill-in of 5=-overhang NHEJ...

RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

“…As such, these clastogens, although time tested and informative, do not provide a specific readout of specific DNA repair pathways. We have adapted the homing endonuclease I-SceI to interrogate DSB repair pathway choice in mycobacteria (1,9,10). This system utilizes a recombination reporter substrate integrated into the chromosome and allows scoring of three DSB repair pathway outcomes: HR, NHEJ, and SSA.…”

“…This parallel is especially relevant for mycobacteria, which implement the same three distinct DSB repair pathways operative in eukarya: homologous recombination (HR), nonhomologous end joining (NHEJ), and singlestrand annealing (SSA). Mycobacterial NHEJ relies on the end-binding protein Ku, DNA ligase LigD (a multifunctional ligase-polymerase-3=-phosphoesterase), DNA ligase LigC1, and two additional polymerases (PolD1 and PolD2) (1)(2)(3)(4)(5)(6)(7)(8)(9). The SSA pathway of mycobacteria repairs DSBs that arise between repeats, with consequent deletion of the DNA between the repeats.…”

Multiple and Variable NHEJ-Like Genes Are Involved in Resistance to DNA Damage in Streptomyces ambofaciens

“…The initial model involving two unique actors is now renewed by the discovery of alternative actors. When the ligase domain of LigD (LigDom) is defective, a back-up ligation activity is provided by LigC, another ATP dependent ligase (Della et al, 2004; Bhattarai et al, 2014). In the same way, Mycobacterium smegmatis possesses two additional polymerases which share the same in vitro activity than the polymerase domain of LigD (PolDom) (Zhu et al, 2012).…”

Ribonucleotides in bacterial DNA

“…The PE domain plays a minor role in NHEJ in vivo , which indicates that this activity is only important for repair at a minority of double-strand breaks (Aniukwu et al, 2008; Bhattarai et al, 2014). Even when active, the PE domain leaves at least one ribonucleotide 3′ to the break, which stimulates the activity of the LigD-LIG domain (Zhu and Shuman, 2008).…”