Non-homologous end joining often uses microhomology: Implications for alternative end joining

Artemis and PALF (also called APLF) appear to be among the primary nucleases involved in non-homologous end joining (NHEJ) and responsible for most nucleolytic end processing in NHEJ. About 60% of NHEJ events show an alignment of the DNA ends that use 1 or 2 bp of microhomology (MH) between the two DNA termini. Thus, MH is a common feature of NHEJ. For most naturally occurring human chromosomal deletions (e.g., after oxidative damage or radiation) and translocations, such as those seen in human neoplasms and as well as inherited chromosomal structural variations, MH usage occurs at a frequency that is typical of NHEJ, and does not suggest major involvement of alternative pathways that require more extensive MH. Though we mainly focus on human NHEJ at double-strand breaks, comparison on these points to other eukaryotes, primarily S. cerevisiae, is informative.     

Control of alternative end joining by the chromatin remodeler p400 ATPase

Repair of DNA double-strand breaks occurs in a chromatin context that needs to be modified and remodeled to allow suitable access to the different DNA repair machineries. Of particular importance for the maintenance of genetic stability is the tight control of error-prone pathways, such as the alternative End Joining pathway. Here, we show that the chromatin remodeler p400 ATPase is a brake to the use of alternative End Joining. Using specific intracellular reporter susbstrates we observed that p400 depletion increases the frequency of alternative End Joining events, and generates large deletions following repair of double-strand breaks. This increase of alternative End Joining events is largely dependent on CtIP-mediated resection, indicating that it is probably related to the role of p400 in late steps of homologous recombination. Moreover, p400 depletion leads to the recruitment of poly(ADP) ribose polymerase (PARP) and DNA ligase 3 at DNA double-strand breaks, driving to selective killing by PARP inhibitors. All together these results show that p400 acts as a brake to prevent alternative End Joining-dependent genetic instability and underline its potential value as a clinical marker.     

Non-homologous DNA end joining

DNA double strand breaks (DSB) are the most serious form of DNA damage. Repair of DSBs is important to prevent chromosomal fragmentation, translocations and deletions. Non-homologous end joining (NHEJ) is one of three major pathways for the repair of DSBs in human cells. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. NHEJ is typically imprecise, a characteristic that is useful for immune diversification in lymphocytes in V(D)J recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku proteins, XRCC4, DNA ligase IV, and Artemis. This review focuses on the mechanisms an dregulation of DSB repair by NHEJ in mammalian cells.     

Modeling non-homologous end joining

Non-homologous end joining (NHEJ) is an important DNA repair pathway for DNA double-strand breaks. Several proteins, including Ku, DNA-PKcs, Artemis, XRCC4/Ligase IV and XLF, are involved in the NHEJ for the DNA damage detection, DNA free end processing and ligation. The classical model of NHEJ is a sequential model in which DNA-PKcs is first recruited by the Ku bound DNA prior to any other repair proteins. Recent experimental study ( [McElhinny et al., 2000], [Costantini et al., 2007], [17] and [Yano and Chen, 2008]) suggested that the recruitment ordering is not crucial. In this work, by proposing a mathematical model in terms of biochemical reaction network and performing stability and related analysis, we demonstrate theoretically that if DSB repair pathway independent of DNA-PKcs exists, then the classical sequential model and new two-phase model are essentially indistinguishable in the sense that DSB can be repaired thoroughly in both models when the repair proteins are sufficient.     

Non-homologous DNA end joining

DNA double-strand breaks (DSBs) are a serious threat for the cell and when not repaired or misrepaired can result in mutations or chromosome rearrangements and eventually in cell death. Therefore, cells have evolved a number of pathways to deal with DSB including homologous recombination (HR), single-strand annealing (SSA) and non-homologous end joining (NHEJ). In mammals DSBs are primarily repaired by NHEJ and HR, while HR repair dominates in yeast, but this depends also on the phase of the cell cycle. NHEJ functions in all kinds of cells, from bacteria to man, and depends on the structure of DSB termini. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. The usage of microhomology is common in DNA end-joining of physiological DSBs, such as at the coding ends in V(D)J (variable(diversity) joining) recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), which is recruited by DNA Ku protein, a heterodimer of Ku70 and Ku80, as well as XRCC4 protein and DNA ligase IV. A complex of Rad50/Mre11/Xrs2, a family of Sir proteins and probably other yet unidentified proteins can be also involved in this process. NHEJ and HR may play overlapping roles in the repair of DSBs produced in the S phase of the cell cycle or at replication forks. Aside from DNA repair, NHEJ may play a role in many different processes, including the maintenance of telomeres and integration of HIV-1 genome into a host genome, as well as the insertion of pseudogenes and repetitive sequences into the genome of mammalian cells. Inhibition of NHEJ can be exploited in cancer therapy in radio-sensitizing cancer cells. Identification of all key players and fundamental mechanisms underlying NHEJ still requires further research.     

The checkpoint clamp protein Rad9 facilitates DNA-end resection and prevents alternative non-homologous end joining

DNA damage activates the cell cycle checkpoint to regulate cell cycle progression. The checkpoint clamp (Rad9-Hus1-Rad1 complex) is recruited to damage sites, and is required for checkpoint activation. While functions of the checkpoint clamp in checkpoint activation have been well studied, its functions in DNA repair regulation remain elusive. Here we show that Rad9 is required for efficient homologous recombination (HR), and facilitates DNA-end resection. The role of Rad9 in homologous recombination is independent of its function in checkpoint activation, and this function is important for preventing alternative non-homologous end joining (altNHEJ). These findings reveal novel function of the checkpoint clamp in HR.     

Malaria parasites utilize both homologous recombination and alternative end joining pathways to maintain genome integrity

Malaria parasites replicate asexually within their mammalian hosts as haploid cells and are subject to DNA damage from the immune response and chemotherapeutic agents that can significantly disrupt genomic integrity. Examination of the annotated genome of the parasite Plasmodium falciparum identified genes encoding core proteins required for the homologous recombination (HR) pathway for repairing DNA double-strand breaks (DSBs), but surprisingly none of the components of the canonical non-homologous end joining (C-NHEJ) pathway were identified. To better understand how malaria parasites repair DSBs and maintain genome integrity, we modified the yeast I-SceI endonuclease system to generate inducible, site-specific DSBs within the parasite’s genome. Analysis of repaired genomic DNA showed that parasites possess both a typical HR pathway resulting in gene conversion events as well as an end joining (EJ) pathway for repair of DSBs when no homologous sequence is available. The products of EJ were limited in number and identical products were observed in multiple independent experiments. The repair junctions frequently contained short insertions also found in the surrounding sequences, suggesting the possibility of a templated repair process. We propose that an alternative end-joining pathway rather than C-NHEJ, serves as a primary method for repairing DSBs in malaria parasites.     

Fanconi anemia-associated chromosomal radial formation is dependent on POLθ-mediated alternative end joining

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Characterization of sequence contexts that favor alternative end joining at Cas9-induced double-strand breaks

Alternative end joining (alt-EJ) mechanisms, such as polymerase theta-mediated end joining, are increasingly recognized as important contributors to inaccurate double-strand break repair. We previously proposed an alt-EJ model whereby short DNA repeats near a double-strand break anneal to form secondary structures that prime limited DNA synthesis. The nascent DNA then pairs with microhomologous sequences on the other break end. This synthesis-dependent microhomology-mediated end joining (SD-MMEJ) explains many of the alt-EJ repair products recovered following I-SceI nuclease cutting in Drosophila. However, sequence-specific factors that influence SD-MMEJ repair remain to be fully characterized. Here, we expand the utility of the SD-MMEJ model through computational analysis of repair products at Cas9-induced double-strand breaks for 1100 different sequence contexts. We find evidence at single nucleotide resolution for sequence characteristics that drive successful SD-MMEJ repair. These include optimal primer repeat length, distance of repeats from the break, flexibility of DNA sequence between primer repeats, and positioning of microhomology templates relative to preferred primer repeats. In addition, we show that DNA polymerase theta is necessary for most SD-MMEJ repair at Cas9 breaks. The analysis described here includes a computational pipeline that can be utilized to characterize preferred mechanisms of alt-EJ repair in any sequence context.     

COM1, a factor of alternative non‐homologous end joining,lagging behind the classic non‐homologous end joining pathway in rice somatic cells

The role of rice (Oryza sativa) COM1 in meiotic homologous recombination (HR) is well understood, but its part in somatic double‐stranded break (DSB) repair remains unclear. Here, we show that for rice plants COM1 conferred tolerance against DNA damage caused by the chemicals bleomycin and mitomycin C, while the COM1 mutation did not compromise HR efficiencies and HR factor (RAD51 and RAD51 paralogues) localization to irradiation‐induced DSBs. Similar retarded growth at the post‐germination stage was observed in the com1‐2 mre11 double mutant and the mre11 single mutant, while combined mutations in COM1 with the HR pathway gene (RAD51C) or classic non‐homologous end joining (NHEJ) pathway genes (KU70, KU80, and LIG4) caused more phenotypic defects. In response to γ‐irradiation, COM1 was loaded normally onto DSBs in the ku70 mutant, but could not be properly loaded in the MRE11^RNAi plant and in the wortmannin‐treated wild‐type plant. Under non‐irradiated conditions, more DSB sites were occupied by factors (MRE11, COM1, and LIG4) than RAD51 paralogues (RAD51B, RAD51C, and XRCC3) in the nucleus of wild‐type; protein loading of COM1 and XRCC3 was increased in the ku70 mutant. Therefore, quite differently to its role for HR in meiocytes, rice COM1 specifically acts in an alternative NHEJ pathway in somatic cells, based on the Mre11–Rad50–Nbs1 (MRN) complex and facilitated by PI3K‐like kinases. NHEJ factors, not HR factors, preferentially load onto endogenous DSBs, with KU70 restricting DSB localization of COM1 and XRCC3 in plant somatic cells.     

Enzymatic end-to end joining of DNA molecules

A way to join naturally occurring DNA molecules, independent of their base sequence, is proposed, based upon the presumed ability of the calf thymus enzyme terminal deoxynucleotidyltransferase to add homopolymer blocks to the ends of double-stranded DNA. To test the proposal, covalently closed dimer circles of the DNA of bacteriophage P22 were produced from linear monomers. It is found that P22 DNA as isolated will prime the terminal transferase reaction, but not in a satisfactory manner. Pre-treatment of the DNA with λ exonuclease, however, improves its priming ability. Terminal transferase can then be used to add oligo(dA) blocks to the ends of one population of P22 DNA molecules and oligo(dT) blocks to the ends of a second population, which enables the two DNAs to anneal to one another to form dimer circles. Subsequent treatment with a system of DNA repair enzymes converts the circles to covalently closed molecules at high efficiency. It is demonstrated that the success of the joining system does not depend upon any obvious unique property of the P22 DNA.The joining system yields several classes of by-products, among them closed circular molecules with branches. Their creation can be explained on the basis of the properties of terminal transferase and the DNA repair enzymes.     

Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining

Exposure of cells to ionizing radiation or radiomimetic drugs generates DNA double-strand breaks that are processed either by homologous recombination repair (HRR), or by canonical, DNA-PKcs-dependent non-homologous end-joining (C-NHEJ). Chemical or genetic inactivation of factors involved in C-NHEJ or HRR, but also their local failure in repair proficient cells, promotes an alternative, error-prone end-joining pathway that serves as backup (A-EJ). There is evidence for the involvement of Artemis endonuclease, a protein deficient in a human radiosensitivity syndrome associated with severe immunodeficiency (RS-SCID), in the processing of subsets of DSBs by HRR or C-NHEJ. It is thought that within HRR or C-NHEJ Artemis processes DNA termini at complex DSBs. Whether Artemis has a role in A-EJ remains unknown. Here, we analyze using pulsed-field gel electrophoresis (PFGE) and specialized reporter assays, DSB repair in wild-type pre-B NALM-6 lymphocytes, as well as in their Artemis^−/−, DNA ligase 4^−/− (LIG4^−/−), and LIG4^−/−/Artemis^−/− double mutant counterparts, under conditions allowing evaluation of A-EJ. Our results substantiate the suggested roles of Artemis in C-NHEJ and HRR, but also demonstrate a role for the protein in A-EJ that is confirmed in Artemis deficient normal human fibroblasts. We conclude that Artemis is a nuclease participating in DSB repair by all major repair pathways.     

Bacterial DNA repair by non-homologous end joining

The capacity to rectify DNA double-strand breaks (DSBs) is crucial for the survival of all species. DSBs can be repaired either by homologous recombination (HR) or non-homologous end joining (NHEJ). The long-standing notion that bacteria rely solely on HR for DSB repair has been overturned by evidence that mycobacteria and other genera have an NHEJ system that depends on a dedicated DNA ligase, LigD, and the DNA-end-binding protein Ku. Recent studies have illuminated the role of NHEJ in protecting the bacterial chromosome against DSBs and other clastogenic stresses. There is also emerging evidence of functional crosstalk between bacterial NHEJ proteins and components of other DNA-repair pathways. Although still a young field, bacterial NHEJ promises to teach us a great deal about the nexus of DNA repair and bacterial pathogenesis.     

The mechanism of human nonhomologous DNA end joining

Double-strand breaks are common in all living cells, and there are two major pathways for their repair. In eukaryotes, homologous recombination is restricted to late S or G(2), whereas nonhomologous DNA end joining (NHEJ) can occur throughout the cell cycle and is the major pathway for the repair of double-strand breaks in multicellular eukaryotes. NHEJ is distinctive for the flexibility of the nuclease, polymerase, and ligase activities that are used. This flexibility permits NHEJ to function on the wide range of possible substrate configurations that can arise when double-strand breaks occur, particularly at sites of oxidative damage or ionizing radiation. NHEJ does not return the local DNA to its original sequence, thus accounting for the wide range of end results. Part of this heterogeneity arises from the diversity of the DNA ends, but much of it arises from the many alternative ways in which the nuclease, polymerases, and ligase can act during NHEJ. Physiologic double-strand break processes make use of the imprecision of NHEJ in generating antigen receptor diversity. Pathologically, the imprecision of NHEJ contributes to genome mutations that arise over time.     

Modulation of DNA end joining by nuclear proteins

DNA double strand breaks in mammalian cells are primarily repaired by homologous recombination and non-homologous end joining (NHEJ). NHEJ may either be error-free or mutagenic with deletions or insertions at the joint. Recent studies showed that DNA ends can also be joined via microhomologous sequences flanking the break point especially when proteins responsible for NHEJ, such as Ku, are absent. Microhomology-mediated end joining (MHEJ) is always accompanied by a deletion that spans one of the two homologous sequences and the intervening sequence, if any. In this study we evaluated several factors affecting the relative contribution of MHEJ to DNA end joining using nuclear extracts and DNA substrates containing 10-bp repeats at the ends. We found that the occurrence of MHEJ is determined by the relative abundance of nuclear proteins. At low DNA/protein ratios, an error-free end-joining mechanism predominated over MHEJ. As the DNA/protein ratio increased, MHEJ became predominant. We show that the nuclear proteins that contribute to the inhibition of the error-prone MHEJ include Ku and histone H1. Treatment of extracts with flap endonuclease 1 antiserum significantly reduced MHEJ. Addition of a 17-bp intervening sequence between the microhomologous sequences significantly reduced the efficiency of MHEJ. Thus, this cell-free assay provides a platform for evaluating factors modulating end joining.     

Interactive competition between homologous recombination and non-homologous end joining

DNA-dependent protein kinase (DNA-PK), composed of Ku70, Ku80, and the catalytic subunit (DNA-PKcs), is involved in double-strand break (DSB) repair by non-homologous end joining (NHEJ). DNA-PKcs defects confer ionizing radiation sensitivity and increase homologous recombination (HR). Increased HR is consistent with passive shunting of DSBs from NHEJ to HR. We therefore predicted that inhibiting the DNA-PKcs kinase would increase HR. A novel DNA-PKcs inhibitor (1-(2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone; designated IC86621) increased ionizing radiation sensitivity but surprisingly decreased spontaneous and DSB-induced HR. Wortmannin also inhibits DNA-PKcs and reduces DSB-induced HR. IC86621 did not affect HR product outcome, indicating that it affects HR initiation. Thus, HR is increased in the absence of DNA-PKcs, but decreased when DNA-PKcs is catalytically inactive, suggesting interactive competition between HR and NHEJ. The effects of IC86621 and wortmannin were proportional to the level of DNA-PKcs, consistent with inhibited DNA-PKcs acting in a dominant negative manner. We propose that inhibition of DNA-PKcs blocks its autophosphorylation, prevents dissociation of DNA-PKcs from DNA ends, and thereby blocks both HR and NHEJ. By blocking the two major DSB repair pathways, DNA-PKcs inhibitors should radiosensitize at all cell-cycle stages and are therefore excellent candidates for augmenting cancer radiotherapy.     

Non-homologous end joining: Emerging themes and unanswered questions

Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks in human cells. Here, we discuss current insights into the mechanism of NHEJ and the interplay between NHEJ and other pathways for repair of IR-induced DNA damage.