Nonhomologous DNA End Joining Pathway Research Articles

Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit.

CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries.

The flexible and iterative steps within the NHEJ pathway

Cellular and biochemical studies of nonhomologous DNA end joining (NHEJ) have long established that nuclease and polymerase action are necessary for the repair of a very large fraction of naturally-arising double-strand breaks (DSBs). This conclusion is derived from NHEJ studies ranging from yeast to humans and all genetically-tractable model organisms. Biochemical models derived from recent real-time and structural studies have yet to incorporate physical space or timing for DNA end processing. In real-time single molecule FRET (smFRET) studies, we analyzed NHEJ synapsis of DNA ends in a defined biochemical system. We described a Flexible Synapsis (FS) state in which the DNA ends were in proximity via only Ku and XRCC4:DNA ligase 4 (X4L4), and in an orientation that would not yet permit ligation until base pairing between one or more nucleotides of microhomology (MH) occurred, thereby allowing an in-line Close Synapsis (CS) state. If no MH was achievable, then XLF was critical for ligation. Neither FS or CS required DNA-PKcs, unless Artemis activation was necessary to permit local resection and subsequent base pairing between the two DNA ends being joined. Here we conjecture on possible 3D configurations for this FS state, which would spatially accommodate the nuclease and polymerase processing steps in an iterative manner. The FS model permits repeated attempts at ligation of at least one strand at the DSB after each round of nuclease or polymerase action. In addition to activation of Artemis, other possible roles for DNA-PKcs are discussed.

Nonhomologous DNA end joining of nucleosomal substrates in a purified system

The nonhomologous DNA end joining pathway is required for repair of most double-strand breaks in the mammalian genome. Here we use a purified biochemical NHEJ system to compare the joining of free DNA with recombinant mononucleosomal and dinucleosomal substrates to investigate ligation and local DNA end resection. We find that the nucleosomal state permits ligation in a manner dependent on the presence of free DNA flanking the nucleosome core particle. Local resection at DNA ends by the Artemis:DNA-PKcs nuclease complex is completely suppressed in all mononucleosome substrates regardless of flanking DNA up to a length of 14 bp. Like mononucleosomes, dinucleosomes lacking flanking free DNA are not joined. Therefore, the nucleosomal state imposes severe constraints on NHEJ nuclease and ligase activities.

The molecular basis and disease relevance of non-homologous DNA end joining.

Non-homologous DNA end joining (NHEJ) is the predominant repair mechanism of any type of DNA double-strand break (DSB) during most of the cell cycle and is essential for the development of antigen receptors. Defects in NHEJ result in sensitivity to ionizing radiation and loss of lymphocytes. The most critical step of NHEJ is synapsis, or the juxtaposition of the two DNA ends of a DSB, because all subsequent steps rely on it. Recent findings show that, like the end processing step, synapsis can be achieved through several mechanisms. In this Review, we first discuss repair pathway choice between NHEJ and other DSB repair pathways. We then integrate recent insights into the mechanisms of NHEJ synapsis with updates on other steps of NHEJ, such as DNA end processing and ligation. Finally, we discuss NHEJ-related human diseases, including inherited disorders and neoplasia, which arise from rare failures at different NHEJ steps.

Two-long terminal repeat (LTR) DNA circles are a substrate for HIV-1 integrase

Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catalyzed by the retroviral integrase. To date, the only substrate described to be involved in this critical reaction is the linear viral DNA produced in reverse transcription. However, during HIV-1 infection, two-long terminal repeat DNA circles (2-LTRcs) are also generated through the ligation of the viral DNA ends by the host cell's nonhomologous DNA end-joining pathway. These DNAs contain all the genetic information required for viral replication, but their role in HIV-1's life cycle remains unknown. We previously showed that both linear and circular DNA fragments containing the 2-LTR palindrome junction can be efficiently cleaved in vitro by recombinant integrases, leading to the formation of linear 3'-processed-like DNA. In this report, using in vitro experiments with purified proteins and DNAs along with DNA endonuclease and in vivo integration assays, we show that this circularized genome can also be efficiently used as a substrate in HIV-1 integrase-mediated integration both in vitro and in eukaryotic cells. Notably, we demonstrate that the palindrome cleavage occurs via a two-step mechanism leading to a blunt-ended DNA product, followed by a classical 3'-processing reaction; this cleavage leads to integrase-dependent integration, highlighted by a 5-bp duplication of the host genome. Our results suggest that 2-LTRc may constitute a reserve supply of HIV-1 genomes for proviral integration.

Understanding the Histone DNA Repair Code: H4K20me2 Makes Its Mark.

Chromatin is a highly compact structure that must be rapidly rearranged in order for DNA repair proteins to access sites of damage and facilitate timely and efficient repair. Chromatin plasticity is achieved through multiple processes, including the posttranslational modification of histone tails. In recent years, the impact of histone posttranslational modification on the DNA damage response has become increasingly well recognized, and chromatin plasticity has been firmly linked to efficient DNA repair. One particularly important histone posttranslational modification process is methylation. Here, we focus on the regulation and function of H4K20 methylation (H4K20me) in the DNA damage response and describe the writers, erasers, and readers of this important chromatin mark as well as the combinatorial histone posttranslational modifications that modulate H4K20me recognition. Finally, we discuss the central role of H4K20me in determining if DNA double-strand breaks (DSB) are repaired by the error-prone, nonhomologous DNA end joining pathway or the error-free, homologous recombination pathway. This review article discusses the regulation and function of H4K20me2 in DNA DSB repair and outlines the components and modifications that modulate this important chromatin mark and its fundamental impact on DSB repair pathway choice. Mol Cancer Res; 16(9); 1335-45. ©2018 AACR.

Programmable DNA repair with CRISPRa/i enhanced homology-directed repair efficiency with a single Cas9

CRISPR systems have been proven as versatile tools for site-specific genome engineering in mammalian species. During the gene editing processes, these RNA-guide nucleases introduce DNA double strand breaks (DSBs), in which non-homologous DNA end joining (NHEJ) dominates the DNA repair pathway, limiting the efficiency of homology-directed repair (HDR), the alternative pathway essential for precise gene targeting. Multiple approaches have been developed to enhance HDR, including chemical compound or RNA interference-mediated inhibition of NHEJ factors, small molecule activation of HDR enzymes, or cell cycle timed delivery of CRISPR complex. However, these approaches face multiple challenges, yet have moderate or variable effects. Here we developed a new approach that programs both NHEJ and HDR pathways with CRISPR activation and interference (CRISPRa/i) to achieve significantly enhanced HDR efficiency of CRISPR-mediated gene editing. The manipulation of NHEJ and HDR pathway components, such as CtIP, CDK1, KU70, KU80, and LIG4, was mediated by catalytically dead guide RNAs (dgRNAs), thus relying on only a single catalytically active Cas9 to perform both CRISPRa/i and precise gene editing. While reprogramming of most DNA repair factors or their combinations tested enhanced HDR efficiency, simultaneously activating CDK1 and repressing KU80 has the strongest effect with increased HDR rate upto an order of magnitude. Doxycycline-induced dgRNA-based CRISPRa/i programming of DNA repair enzymes, as well as viral packaging enabled flexible and tunable HDR enhancement for broader applicability in mammalian cells. Our study provides an effective, flexible, and potentially safer strategy to enhance precise genome modifications, which might broadly impact human gene editing and therapy.

Non-homologous DNA end joining and alternative pathways to double-strand break repair.

DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage because they can result in the loss of large chromosomal regions. In all mammalian cells, DSBs that occur throughout the cell cycle are repaired predominantly by the non-homologous DNA end joining (NHEJ) pathway. Defects in NHEJ result in sensitivity to ionizing radiation and the ablation of lymphocytes. The NHEJ pathway utilizes proteins that recognize, resect, polymerize and ligate the DNA ends in a flexible manner. This flexibility permits NHEJ to function on a wide range of DNA-end configurations, with the resulting repaired DNA junctions often containing mutations. In this Review, we discuss the most recent findings regarding the relative involvement of the different NHEJ proteins in the repair of various DNA-end configurations. We also discuss the shunting of DNA-end repair to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA) and the relevance of these different pathways to human disease.

Cloning, localization and focus formation at DNA damage sites of canine Ku70

Understanding the molecular mechanisms of DNA double-strand break (DSB) repair machinery, specifically non-homologous DNA-end joining (NHEJ), is crucial for developing next-generation radiotherapies and common chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, might play vital roles for regulation of NHEJ activity. The human Ku heterodimer (Ku70/Ku80) is a core NHEJ factor in the NHEJ pathway and is involved in sensing of DSBs. Companion animals, such as canines, have been proposed to be an excellent model for cancer research, including development of chemotherapeutics. However, the post-translational modifications, localization and complex formation of canine Ku70 have not been clarified. Here, we show that canine Ku70 localizes in the nuclei of interphase cells and that it is recruited quickly at laser-microirradiated DSB sites. Structurally, two DNA-PK phosphorylation sites (S6 and S51), an ubiquitination site (K114), two canonical sumoylation consensus motifs, a CDK phosphorylation motif, and a nuclear localization signal (NLS) in the human Ku70 are evolutionarily conserved in canine and mouse species, while the acetylation sites in human Ku70 are partially conserved. Intriguingly, the primary candidate nucleophile (K31) required for 5’dRP/AP lyase activity of human and mouse Ku70 is not conserved in canines, suggesting that canine Ku does not possess this activity. Our findings provide insights into the molecular mechanisms of Ku-dependent NHEJ in a canine model and form a platform for the development of next-generation common chemotherapeutics for human and animal cancers.

Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry.

Chromosomal structure of nuclear DNA is usually maintained by insertion of nucleosomes into preexisting chromatin, both on newly synthesized DNA atreplication forks and at sites of DNA damage. But during retrovirus infection, a histone-free DNA copy of the viral genome is synthesized that must be loaded with nucleosomes de novo. Here, we show that core histones are rapidly loaded onto unintegrated Moloney murine leukemia virus DNAs. Loading of nucleosomes requires nuclear entry, but does not require viral DNA integration. The histones associated with unintegrated DNAs become marked by covalent modifications, with a delay relative to thetime of core histone loading. Expression from unintegrated DNA can be enhanced by modulation of the histone-modifying machinery. The data show that histone loading onto unintegrated DNAs occurs very rapidly after nuclear entry and does not require prior establishment of an integrated provirus.

Structure-Specific nuclease activities of Artemis and the Artemis: DNA-PKcs complex.

Artemis is a vertebrate nuclease with both endo- and exonuclease activities that acts on a wide range of nucleic acid substrates. It is the main nuclease in the non-homologous DNA end-joining pathway (NHEJ). Not only is Artemis important for the repair of DNA double-strand breaks (DSBs) in NHEJ, it is essential in opening the DNA hairpin intermediates that are formed during V(D)J recombination. Thus, humans with Artemis deficiencies do not have T- or B-lymphocytes and are diagnosed with severe combined immunodeficiency (SCID). While Artemis is the only vertebrate nuclease capable of opening DNA hairpins, it has also been found to act on other DNA substrates that share common structural features. Here, we discuss the key structural features that all Artemis DNA substrates have in common, thus providing a basis for understanding how this structure-specific nuclease recognizes its DNA targets.

Sirtuin 6 (SIRT6) rescues the decline of homologous recombination repair during replicative senescence

Genomic instability is a hallmark of aging tissues. Genomic instability may arise from the inefficient or aberrant function of DNA double-stranded break (DSB) repair. DSBs are repaired by homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). HR is a precise pathway, whereas NHEJ frequently leads to deletions or insertions at the repair site. Here, we used normal human fibroblasts with a chromosomally integrated HR reporter cassette to examine the changes in HR efficiency as cells progress to replicative senescence. We show that HR declines sharply with increasing replicative age, with an up to 38-fold decrease in efficiency in presenescent cells relative to young cells. This decline is not explained by a reduction of the number of cells in S/G(2)/M stage as presenescent cells are actively dividing. Expression of proteins involved in HR such as Rad51, Rad51C, Rad52, NBS1, and Sirtuin 6 (SIRT6) diminished with cellular senescence. Supplementation of Rad51, Rad51C, Rad52, and NBS1 proteins, either individually or in combination, did not rescue the senescence-related decline of HR. However, overexpression of SIRT6 in "middle-aged" and presenescent cells strongly stimulated HR repair, and this effect was dependent on mono-ADP ribosylation activity of poly(ADP-ribose) polymerase (PARP1). These results suggest that in aging cells, the precise HR pathway becomes repressed giving way to a more error-prone NHEJ pathway. These changes in the processing of DSBs may contribute to age-related genomic instability and a higher incidence of cancer with age. SIRT6 activation provides a potential therapeutic strategy to prevent the decline in genome maintenance.

Time-Dependent Predominance of Nonhomologous DNA End-Joining Pathways during Embryonic Development in Mice

Repair of DNA double-strand breaks (DSBs) is crucial for maintaining genomic integrity during the successful development of a fertilized egg into a whole organism. To date, the mechanism of DSB repair in postimplantation embryos has been largely unknown. In the present study, using a cell-free repair system derived from the different embryonic stages of mice, we find that canonical nonhomologous end joining (NHEJ), one of the major DSB repair pathways in mammals, is predominant at 14.5 day of embryonic development. Interestingly, all four types of DSBs tested were repaired by ligase IV/XRCC4 and Ku-dependent classical NHEJ. Characterization of end-joined junctions and expression studies further showed evidences for canonical NHEJ. Strikingly, in contrast to the above, we observed noncanonical end joining accompanied by DSB resection, dependent on microhomology and ligase III in 18.5-day embryos. Interestingly, we observed an elevated expression of CtIP, MRE11, and NBS1 at this stage, suggesting that it could act as a switch between classical end joining and microhomology-mediated end joining at later stages of embryonic development. Thus, our results establish for the first time the existence of both canonical and alternative NHEJ pathways during the postimplantation stages of mammalian embryonic development.

Establishment of Hamster Cell Lines with EGFP-Tagged Human XRCC4 and Protection from Low-Dose X-Ray Radiation

In clinical settings, cellular resistance to chemotherapy and radiotherapy is a significant component of tumor treatment failure. The mechanisms underlying the control of localization of DNA repair proteins play a key role in the regulation of DNA repair activity. The DNA repair protein XRCC4, which is a regulator of DNA ligase IV activity, might be a key contributor to not only chemoresistance to anticancer agents, e.g., etoposide, but also radioresistance. However, it remains unclear whether XRCC4, which is a key player in nonhomologous DNA-end-joining (NHEJ), plays a role in low-dose radioresistance. In this study, we confirmed that human XRCC4 tagged with the enhanced green fluorescent protein (EGFP-XRCC4), as well as the DNA damage sensor Ku80 tagged with EGFP, mainly localized in the nuclei and its accumulation at DNA damaged sites began immediately after microirradiation. Moreover, we generated and characterized cell lines expressing EGFP-XRCC4 in XRCC4-deficient cells, i.e., XR-1 cells derived from the Chinese hamster ovary. Our findings showed that XR-1 cells were more sensitive than controls (CHO-K1) to low-dose X-irradiation (<0.5 Gy), whereas the radiosensitive phenotype of XR-1 cells was rescued by the expression of EGFP-XRCC4. We also confirmed that EGFP-XRCC4 expressed stably in XR-1 cells stabilizes DNA ligase IV. Altogether, these cell lines might be useful for the study of not only the dynamics and function of XRCC4, but also the molecular mechanism underlying the cellular resistance via the NHEJ pathway to low-dose radiation in mammalian cells.

The Mechanism of Double-Strand DNA Break Repair by the Nonhomologous DNA End-Joining Pathway

Double-strand DNA breaks are common events in eukaryotic cells, and there are two major pathways for repairing them: homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). The various causes of double-strand breaks (DSBs) result in a diverse chemistry of DNA ends that must be repaired. Across NHEJ evolution, the enzymes of the NHEJ pathway exhibit a remarkable degree of structural tolerance in the range of DNA end substrate configurations upon which they can act. In vertebrate cells, the nuclease, DNA polymerases, and ligase of NHEJ are the most mechanistically flexible and multifunctional enzymes in each of their classes. Unlike repair pathways for more defined lesions, NHEJ repair enzymes act iteratively, act in any order, and can function independently of one another at each of the two DNA ends being joined. NHEJ is critical not only for the repair of pathologic DSBs as in chromosomal translocations, but also for the repair of physiologic DSBs created during variable (diversity) joining [V(D)J] recombination and class switch recombination (CSR). Therefore, patients lacking normal NHEJ are not only sensitive to ionizing radiation (IR), but also severely immunodeficient.

A natural compound, methyl angolensate, induces mitochondrial pathway of apoptosis in Daudi cells

Natural products discovered from medicinal plants have played an important role in the treatment of cancer. In an effort to identify novel small molecules which can affect the proliferation of lymphoma cells, we tested methyl angolensate (MA), a plant derived tetranortriterpenoid, purified from the crude extract of the root callus of Soymida febrifuga commonly known as Indian red wood tree. We have tested MA for its cytotoxic properties on Burkitt's lymphoma cell lines, using various cellular assays. We observed that MA induces cytotoxicity in Daudi cells in a dose-dependent manner using trypan blue, MTT and LDH assays. We find that the treatment with MA led to activation of DNA double-strand break repair proteins including KU70 and KU80, suggesting the activation of nonhomologous DNA end joining pathway in surviving cells. Further, we find that methyl angolensate could induce apoptosis by cell cycle analysis, annexin V-FITC staining, DNA fragmentation and PARP cleavage. Besides, MA treatment led to reactive oxygen species generation and loss of mitochondrial transmembrane potential. These results suggest the activation of mitochondrial pathway of apoptosis. Hence, we identify MA as a potential chemotherapeutic agent against Daudi cells.

The Role of Nonhomologous DNA End Joining, Conservative Homologous Recombination, and Single-Strand Annealing in the Cell Cycle-Dependent Repair of DNA Double-Strand Breaks Induced by H2O2in Mammalian Cells

The purpose of this study was to investigate the cell cycle-dependent role of nonhomologous DNA end joining (NHEJ), conservative homologous recombination (HR), and single-strand annealing (SSA) for the repair of simple DNA double-strand breaks (DSBs) induced by H(2)O(2)-mediated OH radicals in CHO cells. Cells of the cell lines V3 (NHEJ-deficient), irs1SF (HR-deficient) and UV41 (SSA-deficient) and their parental cell line AA8 were exposed to various concentrations of H(2)O(2) in G(1) or S phase of the cell cycle and their colony-forming ability was assayed. In G(1) phase, NHEJ was the most important-if not the only-mechanism to repair H(2)O(2)-mediated DSBs; this was similar to results obtained in a parallel study of more complex DSBs induced by sparsely or densely ionizing radiation. Unlike HR (irs1SF)- and SSA (UV41)-deficient cells, the sensitivity of NHEJ-deficient V3 cells to H(2)O(2) relative to parental AA8 cells in G(1) phase is about 50 times higher compared to 200 kV X rays. This points to a specific role of the catalytic subunit of DNA-PK for efficient NHEJ of H(2)O(2)-mediated DSBs that are located at sites critical for the maintenance of the higher-order structure of cellular DNA, whereas X-ray-induced DSBs are distributed stochastically. Surprisingly, SSA-deficient cells in G(1) phase showed an increased sensitivity to high concentrations of H(2)O(2) relative to the parental wild-type cells and to HR-deficient cells, which may be interpreted in terms of a specific type of H(2)O(2)-induced damage requiring SSA for repair after its transfer into S phase. In S phase, HR is the most important mechanism to repair H(2)O(2)-mediated DSBs, followed by NHEJ. In contrast, the action of error-prone SSA may not be beneficial, since SSA-deficient cells are three times more resistant to H(2)O(2) than wild-type AA8 cells. This is likely due to channeling of DSBs into the error-free HR repair pathway or into the potentially error-prone NHEJ pathway. Cells with or without a defect in DSB repair are considerably more sensitive to H(2)O(2) in S phase compared to G(1) phase. This effect is likely due to the fact that topoisomerase II, which is expressed only in proliferating cells, is a target of H(2)O(2), resulting in enhanced accumulation of DSBs and killing of cells treated in S phase with H(2)O(2). The relative sensitivities to H(2)O(2) differ by orders of magnitude for the four cell lines. This seems to be caused mainly by H(2)O(2)-mediated poisoning of topoisomerase IIalpha rather than by a defect in DSB repair.

Study of Expression of Ku70 and NHEJ in CML Cells

Objective FThe DNA double strand breaks (DSB) in mammalian cells are predominantly repaired by a process called non-homologous DNA end joining (NHEJ). Ku70 played a pivotal role in NHEJ pathway. As a common hematological malignant disease with unique chromosomal translocation t (9;22), chronic myeloid leukemia (CML) can be considered as a paradigm for neoplasias that evolve through a multi-step process. As we reported before, protein Ku70 expressed significantly higher in CML than in normal BM cells. Meanwhile, its expression level in blast phase was markedly higher than that in chronic phase. The present study furthermore aims to investigate the expression of the gene Ku70 in CML cells at different clinical stage and reveal the correlation among the expression of the gene Ku70, the protein Ku70 and BCR-ABL in cells of CML. The NHEJ efficiency to repair DSB in CML was also investigated.Methods: Bone marrow cells were collected from 24 cases of normal adults and 27 cases of de novo diagnosed CML patients. 15 CML patients were in chronic phase and 12 in blast phase. The expression of gene Ku70 was detected by RT-PCR and RQ-RT-PCR. The fusion gene BCR-ABL was detected by TaqMan probe Real Time PCR, using ABL as the internal control. Nucleic extracted proteins were used to determine NHEJ efficiency by an in vitro end-ligation system.Results: The mean level of NHEJ activity of normal BM cells and CML cells were 18.6±13.1% vs 24.8±14.9%, .024. The gene Ku 70 expression in normal BM cells and CML cells were 31.08±8.41 vs 544.63±1185.71 copies/10,000 β-Actin copies, P=0.039. gene Ku 70 expressed significantly higher in blast phase (1103.31±1645.62 copies/10,000 β-Actin copies, P<0.01). There were positive correlations of BCR-ABL when compared with the expression of the gene Ku70 (r=0.573 P=0.002) and with the protein Ku70 (r=0.705 P<0.001) in CML cells. There was also significantly correlations between the expression of the gene Ku70 and the protein Ku70 (r=0. 808, P<0.001).Conclusions: The gene and protein of Ku70 were expressed significantly higher in CML than in normal BM cells with NHEJ activity was also enhanced in CML cells. Meanwhile, Ku70 expression level in acute phase was markedly higher than that in chronic phase. There were significantly correlations among the expression of fusion gene BCR-ABL, the gene Ku70 and the protein Ku70 in cells of CML This Study illustrates DNA instability in CML cells which was mainly damaged by DSB, and this kind of DNA damage was repaired by NHEJ pathway predominantly. NHEJ pathway plays an important role in disease progression of CML.

Mechanistic flexibility as a conserved theme across 3 billion years of nonhomologous DNA end-joining: Table 1.

DNA double-strand breaks (DSB) represent the most deleterious form of DNA damage. Two mechanistically distinctive repair pathways have evolved to mend these breaks in eukaryotes: homologous recombination (HR) and nonhomologous DNA end-joining (NHEJ) (Lieber et al. 2003). HR is restricted to late S or G2 of the cell cycle, whereas NHEJ can function throughout the cell cycle and is the primary repair pathway for DSBs. NHEJ is also distinctive for the mechanistic flexibility of the nucleases, polymerases, and ligases in eukaryotes (Lieber 2007; Lieber et al. 2007). Initially, NHEJ was thought to be restricted to eukaryotes because the best-studied prokaryote, Escherichia coli, has no ability to join DNA ends (if it did, molecular cloning would have had a different history) (Table 1). Only when bioinformatists discovered a distantly diverged Ku-like gene in diverse prokaryotic genomes did researchers begin to realize the existence of a similar NHEJ pathway in bacteria (Aravind and Koonin 2001; Doherty et al. 2001; d’Adda di Fagagna et al. 2003). The bacterial Ku homolog is suspected to form a homodimer with a conjectured structure similar to the ring-shaped eukaryotic Ku heterodimer (Weller et al. 2002). The gene for an ATP-dependent ligase named LigD was often found to be adjacent to the Ku gene on the bacterial chromosome (Aravind and Koonin 2001; Doherty et al. 2001; Weller and Doherty 2001). This linkage between Ku and an ATP-dependent ligase prompted more extensive studies and later defined a bacterial NHEJ pathway. Table 1. Possible corresponding components between NHEJ in prokaryotes and eukaryotes So why do some but not all bacteria retain this DNA repair pathway? Bacterial NHEJ is nonessential under rapid proliferation conditions because HR is active and a duplicate genome is present to provide homology donors (Pitcher et al. 2007a; Shuman and Glickman 2007). However, those bacteria that retain the NHEJ pathway spend much of their life cycle in stationary phase, at which point HR is not available for DSB repair for lack of homology donors. In addition, in nature, desiccation and dry heat are two naturally occurring physical processes that produce substantial numbers of DSBs in bacteria. Hence, bacterial Ku and LigD are present in bacterial species that often form endospores, because during sporulation, NHEJ is efficient for the DSB repair (Pitcher et al. 2007a). In many bacterial species, unlike the eukaryotic NHEJ ligase IV, LigD is a large, multidomain protein that contains three components within a single polypeptide: a polymerase (POL) domain, a phosphoesterase (PE) domain, and a ligase (LIG) domain (Shuman and Glickman 2007). Therefore, it is tempting to suggest that bacterial Ku and LigD are necessary and sufficient to repair all the DSBs generated in vivo (Della et al. 2004). However, in this issue of Genes & Development, the study by Aniukwu et al. (2008) from the Shuman and Glickman laboratories describes extensive genetic studies combined with in vivo DSB repair analysis in Mycobacterium smegmatis and clearly demonstrates the complexity and flexibility of the NHEJ pathway in bacteria. They uncover a faithful NHEJ pathway specifically for 3′ overhang DSB repair, which is Ku- and LigD-independent. Their results also show that the structure of the broken ends determines the pathway and outcome of the DSB repair.

Non-Homologous DNA End Joining in Anticancer Therapy

Non-homologous DNA end joining (NHEJ) is the major pathway for the repair of double-strand breaks (DSBs) in human cells. Proteins involved in NHEJ pathway can become molecular targets in the treatment of cancer. Inhibition of this pathway leads to radio- and chemosensitization of cancer cells. This review will focus on the new therapeutic strategies for NHEJ pathway inhibition and their application in anticancer therapy.