The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Genes & Development (Impact Factor: 10.8). 03/2008; 22(4):512-27. DOI: 10.1101/gad.1631908
Source: PubMed


Mycobacteria can repair DNA double-strand breaks (DSBs) via a nonhomologous end-joining (NHEJ) system that includes a dedicated DNA ligase (LigD) and the DNA end-binding protein Ku. Here we exploit an improved plasmid-based NHEJ assay and a collection of Mycobacterium smegmatis strains bearing deletions or mutations in Ku or the DNA ligases to interrogate the contributions of LigD's three catalytic activities (polymerase, ligase, and 3' phosphoesterase) and structural domains (POL, LIG, and PE) to the efficiency and molecular outcomes of NHEJ in vivo. By analyzing in parallel the repair of blunt, 5' overhang, and 3' overhang DSBs, we discovered a novel end-joining pathway specific to breaks with 3' overhangs that is Ku- and LigD-independent and perfectly faithful. This 3' overhang NHEJ pathway is independent of ligases B and C; we surmise that it relies on NAD(+)-dependent LigA, the essential replicative ligase. We find that efficient repair of blunt and 5' overhang DSBs depends stringently on Ku and the LigD POL domain, but not on the LigD polymerase activity, which mainly serves to promote NHEJ infidelity. The lack of an effect of PE-inactivating LigD mutations on NHEJ outcomes, especially the balance between deletions and insertions at blunt or 5' overhang breaks, argues against LigD being the catalyst of deletion formation. Ligase-inactivating LigD mutations (or deletion of the LIG domain) have a modest impact on the efficiency of blunt and 5' overhang DSB repair, because the strand sealing activity can be provided in trans by one of the other resident ATP-dependent ligases (likely LigC). Reliance on the backup ligase is accompanied by a drastic loss of fidelity during blunt end and 5' overhang DSB repair. We conclude that the mechanisms of mycobacterial NHEJ are many and the outcomes depend on the initial structures of the DSBs and the available ensemble of end-processing and end-sealing components, which are not limited to Ku and LigD.

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    • "The resulting repair products exhibited deletions and were strikingly characterized by a strong dependence on short homologous sequences at the junctions [22]. Similar NHEJindependent end joining activities were also found in mycobacteria [24], Arabidopsis [25], Caenorhabditis elegans [26], Xenopus [27], chicken [28], as well as in rodent [29] [30] [31] [32] and human cells [32] [33] [34]. Furthermore, when analyzed, the junctions consistently exhibited a greater use of microhomology (MH), compared to NHEJ. "
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    ABSTRACT: To cope with DNA double strand break (DSB) genotoxicity, cells have evolved two main repair pathways: homologous recombination which uses homologous DNA sequences as repair templates, and non-homologous Ku-dependent end-joining involving direct sealing of DSB ends by DNA ligase IV (Lig4). During the last two decades a third player most commonly named alternative end-joining (A-EJ) has emerged, which is defined as any Ku- or Lig4-independent end-joining process. A-EJ increasingly appears as a highly error-prone bricolage on DSBs and despite expanding exploration, it still escapes full characterization. In the present review, we discuss the mechanism and regulation of A-EJ as well as its biological relevance under physiological and pathological situations, with a particular emphasis on chromosomal instability and cancer. Whether or not it is a genuine DSB repair pathway, A-EJ is emerging as an important cellular process and understanding A-EJ will certainly be a major challenge for the coming years.
    DNA repair 05/2014; 17. DOI:10.1016/j.dnarep.2014.02.007 · 3.11 Impact Factor
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    • "We and others have shown that, in addition to HR, mycobacteria possess a prototypical non-homologous ends joining (NHEJ) apparatus encoded by evolutionarily conserved ku and ligD genes [5]–[9], as well as a single-strand annealing (SSA) pathway [10]. In the NHEJ process, Ku protein binds to the DNA ends and subsequently interacts with multifunctional LigD, which covalently joins together broken DNA strands [11]. Both HR and NHEJ systems have complementary roles in repairing DSBs, but act independently [12], [13]. "
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    ABSTRACT: The intracellular pathogen Mycobacterium tuberculosis (Mtb) is constantly exposed to a multitude of hostile conditions and is confronted by a variety of potentially DNA-damaging assaults in vivo, primarily from host-generated antimicrobial toxic radicals. Exposure to reactive nitrogen species and/or reactive oxygen species causes different types of DNA damage, including oxidation, depurination, methylation and deamination, that can result in single- or double-strand breaks (DSBs). These breaks affect the integrity of the whole genome and, when left unrepaired, can lead to cell death. Here, we investigated the role of the DSB repair pathways, homologous recombination (HR) and non-homologous ends joining (NHEJ), in the survival of Mtb inside macrophages. To this end, we constructed Mtb strains defective for HR (ΔrecA), NHEJ [Δ(ku,ligD)], or both DSB repair systems [Δ(ku,ligD,recA)]. Experiments using these strains revealed that either HR or NHEJ is sufficient for the survival and propagation of tubercle bacilli inside macrophages. Inhibition of nitric oxide or superoxide anion production with L-NIL or apocynin, respectively, enabled the Δ(ku,ligD,recA) mutant strain lacking both systems to survive intracellularly. Complementation of the Δ(ku,ligD,recA) mutant with an intact recA or ku-ligD rescued the ability of Mtb to propagate inside macrophages.
    PLoS ONE 03/2014; 9(3):e92799. DOI:10.1371/journal.pone.0092799 · 3.23 Impact Factor
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    • "C-NHEJ and A-EJ have also been described in bacteria [65]. In Mycobacterium smegmatis, the vast majority of Ku-independent junctions harbor microhomology-mediated deletions, indicating that A-EJ substituted for C-NHEJ during DSB repair [66]. Ku and ligase D are absent in the classical bacterial model Escherichia coli, and A-EJ is the most active end-joining pathway in this species [67]. "
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    ABSTRACT: DNA double-strand breaks (DSBs) are harmful lesions leading to genomic instability or diversity. Non-homologous end-joining (NHEJ) is a prominent DSB repair pathway, which has long been considered to be error-prone. However, recent data have pointed to the intrinsic precision of NHEJ. Three reasons can account for the apparent fallibility of NHEJ: 1) the existence of a highly error-prone alternative end-joining process; 2) the adaptability of canonical C-NHEJ (Ku- and Xrcc4/ligase IV-dependent) to imperfect complementary ends; and 3) the requirement to first process chemically incompatible DNA ends that cannot be ligated directly. Thus, C-NHEJ is conservative but adaptable, and the accuracy of the repair is dictated by the structure of the DNA ends rather than by the C-NHEJ machinery. We present data from different organisms that describe the conservative/versatile properties of C-NHEJ. The advantages of the adaptability/versatility of C-NHEJ are discussed for the development of the immune repertoire and the resistance to ionizing radiation, especially at low doses, and for targeted genome manipulation.
    PLoS Genetics 01/2014; 10(1):e1004086. DOI:10.1371/journal.pgen.1004086 · 7.53 Impact Factor
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