APLF (C2orf13) facilitates nonhomologous end-joining and undergoes ATM-dependent hyperphosphorylation following ionizing radiation

Nonhomologous end-joining (NHEJ) is the major mammalian DNA double-strand break (DSB) repair pathway of DSBs induced by DNA damaging agents. NHEJ is initiated by the recognition of DSBs by the DNA end-binding heterodimer, Ku, and the final step of DNA end-joining is accomplished by the XRCC4-DNA ligase IV complex. We demonstrate that Aprataxin and PNK-like factor (APLF), an endo/exonuclease with an FHA domain and unique zinc fingers (ZFs), interacts with both Ku and XRCC4-DNA ligase IV in human cells. The interaction of APLF with XRCC4-DNA ligase IV is FHA- and phospho-dependent, and is mediated by CK2 phosphorylation of XRCC4 in vitro. In contrast, APLF associates with Ku independently of the FHA and ZF domains, and APLF complexes with Ku at DNA ends. APLF undergoes ionizing radiation (IR) induced ATM-dependent hyperphosphorylation at serine residue 116, which is highly conserved across mammalian APLF homologues. We demonstrate further that depletion of APLF in human cells by siRNA is associated with impaired NHEJ. Collectively, these results suggest that APLF is an ATM target that is involved in NHEJ and facilitates DSB repair, likely via interactions with Ku and XRCC4-DNA ligase IV.     

Pol3 is involved in nonhomologous end-joining in Saccharomyces cerevisiae

Nonhomologous end joining connects DNA ends in the absence of extended sequence homology and requires removal of mismatched DNA ends and gap-filling synthesis prior to a religation step. Pol4 within the Pol X family is the only polymerase known to be involved in end processing during nonhomologous end joining in yeast. The Saccharomyces cerevisiae POL3/CDC2 gene encodes polymerase delta that is involved in DNA replication and other DNA repair processes. Here, we show that POL3 is involved in nonhomologous end joining using a plasmid-based end-joining assay in yeast, in which the pol3-t mutation caused a 1.9- to 3.2-fold decrease in the end-joining efficiency of partially compatible 5' or 3' ends, or incompatible ends, similar to the pol4 mutant. The pol3-t pol4 double mutation showed a synergistic decrease in the efficiency of NHEJ with partially compatible 5' ends or incompatible ends. Sequence analysis of the rejoined junctions recovered from the wild-type cells and mutants indicated that POL3 is required for gap filling at 3' overhangs, but not 5' overhangs during POL4-independent nonhomologous end joining. We also show that either Pol3 or Pol4 is required for simple religation of compatible or blunt ends. These results suggest that Pol3 has a generalized function in end joining in addition to its role in gap filling at 3' overhangs to enhance the overall efficiency of nonhomologous end joining. Moreover, the decreased end-joining efficiency seen in the pol3-t mutant was not due to S-phase arrest associated with the mutant. Taken together, our genetic evidence supports a novel role of Pol3 in nonhomologous end joining that facilitates gap filling at 3' overhangs in the absence of Pol4 to maintain genomic integrity.     

Crystal structure and nonhomologous end-joining function of the ligase component of Mycobacterium DNA ligase D

DNA ligase D (LigD) is a large polyfunctional enzyme involved in nonhomologous end-joining (NHEJ) in mycobacteria. LigD consists of a C-terminal ATP-dependent ligase domain fused to upstream polymerase and phosphoesterase modules. Here we report the 2.4 angstroms crystal structure of the ligase domain of Mycobacterium LigD, captured as the covalent ligase-AMP intermediate with a divalent metal in the active site. A chloride anion on the protein surface coordinated by the ribose 3'-OH and caged by arginine and lysine side chains is a putative mimetic of the 5'-phosphate at a DNA nick. Structure-guided mutational analysis revealed distinct requirements for the adenylylation and end-sealing reactions catalyzed by LigD. We found that a mutation of Mycobacterium LigD that ablates only ligase activity results in decreased fidelity of NHEJ in vivo and a strong bias of mutagenic events toward deletions instead of insertions at the sealed DNA ends. This phenotype contrasts with the increased fidelity of double-strand break repair in deltaligD cells or in a strain in which only the polymerase function of LigD is defective. We surmise that the signature error-prone quality of bacterial NHEJ in vivo arises from a dynamic balance between the end-remodeling and end-sealing steps.     

XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining

DNA nonhomologous end-joining (NHEJ) is a predominant pathway of DNA double-strand break repair in mammalian cells, and defects in it cause radiosensitivity at the cellular and whole-organism levels. Central to NHEJ is the protein complex containing DNA Ligase IV and XRCC4. By searching for additional XRCC4-interacting factors, we identified a previously uncharacterized 33 kDa protein, XRCC4-like factor (XLF, also named Cernunnos), that has weak sequence homology with XRCC4 and is predicted to display structural similarity to XRCC4. We show that XLF directly interacts with the XRCC4-Ligase IV complex in vitro and in vivo and that siRNA-mediated downregulation of XLF in human cell lines leads to radiosensitivity and impaired NHEJ. Furthermore, we establish that NHEJ-deficient 2BN cells derived from a radiosensitive and immune-deficient patient lack XLF due to an inactivating frameshift mutation in its gene, and that reintroduction of wild-type XLF into such cells corrects their radiosensitivity and NHEJ defects. XLF thus constitutes a novel core component of the mammalian NHEJ apparatus.     

Interplay between Cernunnos-XLF and nonhomologous end-joining proteins at DNA ends in the cell

Cernunnos-XLF is the most recently identified core component in the nonhomologous end-joining (NHEJ) pathway for the repair of DNA double strand breaks (DSBs) in mammals. It associates with the XRCC4/ligase IV ligation complex and stimulates its activity in a still unknown manner. NHEJ also requires the DNA-dependent protein kinase that contains a Ku70/Ku80 heterodimer and the DNA-dependent protein kinase catalytic subunit. To understand the interplay between Cernunnos-XLF and the other proteins implicated in the NHEJ process, we have analyzed the interactions of Cernunnos-XLF and NHEJ proteins in cells after treatment with DNA double strand-breaking agents by means of a detergent-based cellular fractionation protocol. We report that Cernunnos-XLF is corecruited with the core NHEJ components on chromatin damaged with DSBs in human cells and is phosphorylated by the DNA-dependent protein kinase catalytic subunit. Our data show a pivotal role for DNA ligase IV in the NHEJ ligation complex assembly and recruitment to DSBs because the association of Cernunnos-XLF with the XRCC4/ligase IV complex relies primarily on the DNA ligase IV component, and an intact XRCC4/ligase IV complex is necessary for Cernunnos-XLF mobilization to damaged chromatin. Conversely, a Cernunnos-XLF defect has no apparent impact on the XRCC4/ligase IV association and recruitment to the DSBs or on the stimulation of the DNA-dependent protein kinase on DNA ends.     

Essential constituents of the 3'-phosphoesterase domain of bacterial DNA ligase D, a nonhomologous end-joining enzyme

DNA ligase D (LigD) catalyzes end-healing and end-sealing steps during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal 3'-phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at a duplex primer-template with a short 3'-ribonucleotide tract. The phosphodiesterase, which cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus, requires the vicinal 2'-OH of the penultimate ribose. The phosphomonoesterase converts the terminal ribonucleoside 3'-PO4 to a 3'-OH. Here we show that the PE domain has a 3'-phosphatase activity on an all-DNA primer-template, signifying that the phosphomonoesterase reaction does not depend on a 2'-OH. The distinctions between the phosphodiesterase and phosphomonoesterase activities are underscored by the results of alanine-scanning, limited proteolysis, and deletion analysis, which show that the two reactions depend on overlapping but nonidentical ensembles of protein functional groups, including: (i) side chains essential for both ribonuclease and phosphatase activity (His-42, His-48, Asp-50, Arg-52, His-84, and Tyr-88); (ii) side chains important for 3'-phosphatase activity but not for 3' ribonucleoside removal (Arg-14, Asp-15, Glu-21, Gln-40, and Glu-82); and (iii) side chains required selectively for the 3'-ribonuclease (Lys-66 and Arg-76). These constellations of critical residues are unique to LigD-like proteins, which we propose comprise a new bifunctional phosphoesterase family.     

Hierarchy of nonhomologous end-joining, single-strand annealing and gene conversion at site-directed DNA double-strand breaks

In mammalian cells, DNA double-strand breaks (DSBs) are repaired by three pathways, nonhomologous end-joining (NHEJ), gene conversion (GC) and single-strand annealing (SSA). These pathways are distinct with regard to repair efficiency and mutagenic potential and must be tightly controlled to preserve viability and genomic stability. Here, we employed chromosomal reporter constructs to characterize the hierarchy of NHEJ, GC and SSA at a single I-SceI-induced DSB in Chinese hamster ovary cells. We discovered that the use of GC and SSA was increased by 6- to 8-fold upon loss of Ku80 function, suggesting that NHEJ is dominant over the other two pathways. However, NHEJ efficiency was not altered if GC was impaired by Rad51 knockdown. Interestingly, when SSA was made available as an alternative mode for DSB repair, loss of Rad51 function led to an increase in SSA activity at the expense of NHEJ, implying that Rad51 may indirectly promote NHEJ by limiting SSA. We conclude that a repair hierarchy exists to limit the access of the most mutagenic mechanism, SSA, to the break site. Furthermore, the cellular choice of repair pathways is reversible and can be influenced at the level of effector proteins such as Ku80 or Rad51.     

Measuring nonhomologous end-joining,homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.     

Effects of DNA nonhomologous end-joining factors on telomere length and chromosomal stability in mammalian cells

DNA repair by nonhomologous end-joining (NHEJ) relies on the Ku70:Ku80 heterodimer in species ranging from yeast to man. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, Ku also controls telomere functions. Here, we show that Ku70, Ku80, and DNA-PKcs, with which Ku interacts, associate in vivo with telomeric DNA in several human cell types, and we show that these associations are not significantly affected by DNA-damaging agents. We also demonstrate that inactivation of Ku80 or Ku70 in the mouse yields telomeric shortening in various primary cell types at different developmental stages. By contrast, telomere length is not altered in cells impaired in XRCC4 or DNA ligase IV, two other NHEJ components. We also observe higher genomic instability in Ku-deficient cells than in XRCC4-null cells. This suggests that chromosomal instability of Ku-deficient cells results from a combination of compromised telomere stability and defective NHEJ.     

Impaired nonhomologous end-joining provokes soft tissue sarcomas harboring chromosomal translocations, amplifications, and deletions.

Although nonhomologous end-joining (NHEJ) deficiency has been shown to accelerate lymphoma formation in mice, its role in suppressing tumors in cells that do not undergo V(D)J recombination is unclear. Utilizing a tumor-prone mouse strain (ink4a/arf(-/-)), we examined the impact of haploinsufficiency of a NHEJ component, DNA ligase IV (Lig4), on murine tumorigenesis. We demonstrate that lig4 heterozygosity promotes the development of soft-tissue sarcomas that possess clonal amplifications, deletions, and translocations. That these genomic alterations are relevant in tumorigenesis is supported by the finding of frequent mdm2 amplification, a known oncogene in human sarcoma. Together, these findings support the view that loss of a single lig4 allele results in NHEJ activity being sufficiently reduced to engender chromosomal aberrations that drive non-lymphoid tumorigenesis.     

Frameshift mutagenesis: the roles of primer-template misalignment and the nonhomologous end-joining pathway in Saccharomyces cerevisiae

Small insertions or deletions that alter the reading frame of a gene typically occur in simple repeats such as mononucleotide runs and are thought to reflect spontaneous primer-template misalignment during DNA replication. The resulting extrahelical repeat is efficiently recognized by the mismatch repair machinery, which specifically replaces the newly replicated strand to restore the original sequence. Frameshift mutagenesis is most easily studied using reversion assays, and previous studies in Saccharomyces cerevisiae suggested that the length threshold for polymerase slippage in mononucleotide runs is 4N. Because the probability of slippage is strongly correlated with run length, however, it was not clear whether shorter runs were unable to support slippage or whether the resulting frameshifts were obscured by the presence of longer runs. To address this issue, we removed all mononucleotide runs >3N from the yeast lys2ΔBgl and lys2ΔA746 frameshift reversion assays, which detect net 1-bp deletions and insertions, respectively. Analyses demonstrate that 2N and 3N runs can support primer-template misalignment, but there is striking run-specific variation in the frequency of slippage, in the accumulation of +1 vs. -1 frameshifts and in the apparent efficiency of mismatch repair. We suggest that some of this variation reflects the role of flanking sequence in initiating primer-template misalignment and that some reflects replication-independent frameshifts generated by the nonhomologous end-joining pathway. Finally, we demonstrate that nonhomologous end joining is uniquely required for the de novo creation of tandem duplications from noniterated sequence.     

Processing of DNA for nonhomologous end-joining is controlled by kinase activity and XRCC4/ligase IV

Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. To repair the broken ends, NHEJ processes noncompatible ends into a ligatable form but suppresses processing of compatible ends. It is not known how NHEJ controls polymerase and nuclease activities to act exclusively on noncompatible ends. Here, we analyzed processing independently of ligation by using a two-stage assay with extracts that recapitulated the properties of NHEJ in vivo. Processing of noncompatible ends required wortmannin-sensitive kinase activity. Since DNA-dependent protein kinase catalytic subunit (DNA-PKcs) brings the ends together before undergoing activation of its kinase, this suggests that processing occurred after synapsis of the ends. Surprisingly, all polymerase and most nuclease activity required XRCC4/Ligase IV. This suggests a mechanism for how NHEJ suppresses processing to optimize the preservation of DNA sequence.     

Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination

Nonhomologous end joining (NHEJ) eliminates DNA double-strand breaks (DSBs) in bacteria and eukaryotes. In Saccharomyces cerevisiae, there are pairwise physical interactions among the core complexes of the NHEJ pathway, namely Yku70-Yku80 (Ku), Dnl4-Lif1 and Mre11-Rad50-Xrs2 (MRX). However, MRX also has a key role in the repair of DSBs by homologous recombination (HR). Here we have examined the assembly of NHEJ complexes at DSBs biochemically and by chromatin immunoprecipitation. Ku first binds to the DNA end and then recruits Dnl4-Lif1. Notably, Dnl4-Lif1 stabilizes the binding of Ku to in vivo DSBs. Ku and Dnl4-Lif1 not only initiate formation of the nucleoprotein NHEJ complex but also attenuate HR by inhibiting DNA end resection. Therefore, Dnl4-Lif1 plays an important part in determining repair pathway choice by participating at an early stage of DSB engagement in addition to providing the DNA ligase activity that completes NHEJ.     

Cernunnos interacts with the XRCC4 x DNA-ligase IV complex and is homologous to the yeast nonhomologous end-joining factor Nej1

DNA double strand breaks are considered as the most harmful DNA lesions and are repaired by either homologous recombination or nonhomologous end joining (NHEJ). A new NHEJ factor, Cernunnos, has been identified, the defect of which leads to a severe immunodeficiency condition associated with microcephaly and other developmental defects in humans. This presentation is reminiscent to that of DNA-ligase IV deficiency and suggests a possible interplay between Cernunnos and the XRCC4 x DNA-ligase IV complex. We show here that Cernunnos physically interacts with the XRCC4 x DNA-ligase IV complex. Moreover, a combination of sensitive methods of sequence analysis revealed that Cernunnos can be associated with the XRCC4 family of proteins and that it corresponds to the genuine homolog of the yeast Nej1 protein. Altogether these results shed new lights on the last step, the DNA religation, of the NHEJ pathway.     

Notch2 and Notch3 function together to regulate vascular smooth muscle development

Notch signaling has been implicated in the regulation of smooth muscle differentiation, but the precise role of Notch receptors is ill defined. Although Notch3 receptor expression is high in smooth muscle, Notch3 mutant mice are viable and display only mild defects in vascular patterning and smooth muscle differentiation. Notch2 is also expressed in smooth muscle and Notch2 mutant mice show cardiovascular abnormalities indicative of smooth muscle defects. Together, these findings infer that Notch2 and Notch3 act together to govern vascular development and smooth muscle differentiation. To address this hypothesis, we characterized the phenotype of mice with a combined deficiency in Notch2 and Notch3. Our results show that when Notch2 and Notch3 genes are simultaneously disrupted, mice die in utero at mid-gestation due to severe vascular abnormalities. Assembly of the vascular network occurs normally as assessed by Pecam1 expression, however smooth muscle cells surrounding the vessels are grossly deficient leading to vascular collapse. In vitro analysis show that both Notch2 and Notch3 robustly activate smooth muscle differentiation genes, and Notch3, but not Notch2 is a target of Notch signaling. These data highlight the combined actions of the Notch receptors in the regulation of vascular development, and suggest that while these receptors exhibit compensatory roles in smooth muscle, their functions are not entirely overlapping.     

Top and bottom yeasts together accelerate ethanol production in molasses fermentation

Summary Alcohol producing top and bottom yeasts were employed individually and together to assess their role in enhancing the rate of ethanol production, in cane molasses fermentation, at 30°C. The combination of top yeastS.cerevisiae NCIM 3281, and bottom yeastS.uvarum NCIM 3509, improved the enthanol production rate by 32.6% in batch fermentation and 25.2% in recycling yeasts cell fermentation as compared to their mean value of individual ethanol production activity.     

Hypersensitivity of nonhomologous DNA end-joining mutants to VP-16 and ICRF-193: implications for the repair of topoisomerase II-mediated DNA damage

A number of clinically useful anticancer drugs, including etoposide (VP-16), target DNA topoisomerase (topo) II. These drugs, referred to as topo II poisons, stabilize cleavable complexes, thereby generating DNA double-strand breaks. Bis-2,6-dioxopiperazines such as ICRF-193 also inhibit topo II by inducing a distinct type of DNA damage, termed topo II clamps, which has been believed to be devoid of double-strand breaks. Despite the biological and clinical importance, the molecular mechanisms for the repair of topo II-mediated DNA damage remain largely unknown. Here, we perform genetic analyses using the chicken DT40 cell line to investigate how DNA lesions caused by topo II inhibitors are repaired. Notably, we show that LIG4-/- and KU70-/- cells, which are defective in nonhomologous DNA end-joining (NHEJ), are extremely sensitive to both VP-16 and ICRF-193. In contrast, RAD54-/- cells (defective in homologous recombination) are much less hypersensitive to VP-16 than the NHEJ mutants and, more importantly, are not hypersensitive to ICRF-193. Our results provide the first evidence that NHEJ is the predominant pathway for the repair of topo II-mediated DNA damage; that is, cleavable complexes and topo II clamps. The outstandingly increased cytotoxicity of topo II inhibitors in the absence of NHEJ suggests that simultaneous inhibition of topo II and NHEJ would provide a powerful protocol in cancer chemotherapy involving topo II inhibitors.     

Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end joining (NHEJ) that closely resemble events in mammalian cells. In one pathway the chromosome ends are degraded to yield deletions with different sizes whose endpoints have 1 to 6 bp of homology. Alternatively, the 4-bp overhanging 3' ends of HO-cut DNA (5'-AACA-3') are not degraded but can be base paired in misalignment to produce +CA and +ACA insertions. When HO was expressed throughout the cell cycle, the efficiency of NHEJ repair was 30 times higher than when HO was expressed only in G1. The types of repair events were also very different when HO was expressed throughout the cell cycle; 78% of survivors had small insertions, while almost none had large deletions. When HO expression was confined to the G1 phase, only 21% were insertions and 38% had large deletions. These results suggest that there are distinct mechanisms of NHEJ repair producing either insertions or deletions and that these two pathways are differently affected by the time in the cell cycle when HO is expressed. The frequency of NHEJ is unaltered in strains from which RAD1, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, deletions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold when HO was not cell cycle regulated. Moreover, mutations in these three genes markedly reduced +CA insertions, while significantly increasing the proportion of both small (-ACA) and larger deletion events. In contrast, the rad5O mutation had little effect on the viability of G1-induced cells but significantly reduced the frequency of both +CA insertions and -ACA deletions in favor of larger deletions. Thus, RAD50 (and by extension XRS2 and MRE11) exerts a much more important role in the insertion-producing pathway of NHEJ repair found in S and/or G2 than in the less frequent deletion events that predominate when HO is expressed only in G1.     

Human induced pluripotent cells resemble embryonic stem cells demonstrating enhanced levels of DNA repair and efficacy of nonhomologous end-joining

To maintain the integrity of the organism, embryonic stem cells (ESC) need to maintain their genomic integrity in response to DNA damage. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and can have disastrous consequences if not repaired correctly, leading to cell death, genomic instability and cancer. How human ESC (hESC) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. Adult somatic cells can be induced to "dedifferentiate" into induced pluripotent stem cells (iPSC) and reprogram into cells of all three germ layers. Whether iPSC have reprogrammed the DNA damage response is a critical question in regenerative medicine. Here, we show that hESC demonstrate high levels of endogenous reactive oxygen species (ROS) which can contribute to DNA damage and may arise from high levels of metabolic activity. To potentially counter genomic instability caused by DNA damage, we find that hESC employ two strategies: First, these cells have enhanced levels of DNA repair proteins, including those involved in repair of DSBs, and they demonstrate elevated nonhomologous end-joining (NHEJ) activity and repair efficacy, one of the main pathways for repairing DSBs. Second, they are hypersensitive to DNA damaging agents, as evidenced by a high level of apoptosis upon irradiation. Importantly, iPSC, unlike the parent cells they are derived from, mimic hESC in their ROS levels, cell cycle profiles, repair protein expression and NHEJ repair efficacy, indicating reprogramming of the DNA repair pathways. Human iPSC however show a partial apoptotic response to irradiation, compared to hESC. We suggest that DNA damage responses may constitute important markers for the efficacy of iPSC reprogramming.     

Mechanism of nonhomologous end-joining in mycobacteria: a low-fidelity repair system driven by Ku, ligase D and ligase C

DNA double-strand breaks (DSBs) can be repaired either via homologous recombination (HR) or nonhomologous end-joining (NHEJ). Both pathways are operative in eukaryotes, but bacteria had been thought to rely on HR alone. Here we provide direct evidence that mycobacteria have a robust NHEJ pathway that requires Ku and a specialized polyfunctional ATP-dependent DNA ligase (LigD). NHEJ of blunt-end and complementary 5'-overhang DSBs is highly mutagenic ( approximately 50% error rate). Analysis of the recombination junctions ensuing from individual NHEJ events highlighted the participation of several DNA end-remodeling activities, including template-dependent fill-in of 5' overhangs, nontemplated addition of single nucleotides at blunt ends, and nucleolytic resection. LigD itself has the template-dependent and template-independent polymerase functions in vitro that compose the molecular signatures of NHEJ in vivo. Another ATP-dependent DNA ligase (LigC) provides a backup mechanism for LigD-independent error-prone repair of blunt-end DSBs. We speculate that NHEJ allows mycobacteria to evade genotoxic host defense.