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.     

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.     

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.     

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.     

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.     

Nonhomologous DNA end joining in cell-free systems.

Double strand DNA breaks are usually caused by ionizing radiation and radiomimetic drugs, but can also occur under normal physiological conditions during double strand break-induced recombination, such as the rearrangement of T-cell receptor and immunoglobulin genes during lymphoid development or the mating type switching in yeast. The main repair mechanism for double strand breaks in higher eukaryotes is nonhomologous DNA end joining (NHEJ), which modifies and ligates the two DNA ends without the help of extensive base-pairing interactions for alignment. Defects in double strand break repair are associated with radiosensitivity, predisposition to cancer and immunodeficiency syndromes, and the analysis of the underlying mutations has lead to the identification of several proteins involved in NHEJ. However, these genetic studies have yielded little information on the mechanism of NHEJ, and while some of the protein factors identified possess the expected enzymatic or DNA-binding activities, the precise role of others remains unclear. Systems for cell-free NHEJ have been available for over 10 years, but the biochemical analysis of NHEJ has lagged behind the genetic analysis, and not a single protein factor required for NHEJ has been identified by biochemical purification and reconstitution of NHEJ activity. Here I review the current status of in vitro systems for NHEJ, summarize the results obtained and information gained, and discuss the outlook for biochemical approaches to study NHEJ.     

Generation of telomeric G strand overhangs involves both G and C strand cleavage

Processing of telomeric DNA is required to generate the 3' G strand overhangs necessary for capping chromosome ends. We have investigated the steps involved in telomere processing by examining G overhang structure in Tetrahymena cells that lack telomerase or have altered telomeric sequences. We show that overhangs are generated by two precise cleavage steps involving nucleases that are robust but lack sequence specificity. Our data suggest that a G overhang binding protein delineates the boundaries for G and C strand cleavage. We also show that telomerase is not the nuclease responsible for G strand cleavage, although telomerase depletion alters the precision of processing. This change in processing indicates that telomerase affects multiple transactions at the telomere and provides a physical footprint for the continued association of telomerase with the telomere after repeat addition is complete.     

Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining

Nonhomologous end joining (NHEJ) directly rejoins DNA double-strand breaks (DSBs) when recombination is not possible. In Saccharomyces cerevisiae, the DNA polymerase Pol4 is required for gap filling when a short 3' overhang must prime DNA synthesis. Here, we examined further end variations to test specific hypotheses regarding Pol4 usage in NHEJ in vivo. Surprisingly, Pol4 dependence at 3' overhangs was reduced when a nonhomologous 5' flap nucleotide was present across from the gap, even though the mismatched nucleotide was corrected, not incorporated. In contrast, a gap with a 5' deoxyribosephosphate (dRP) was as Pol4-dependent as a gap with a 5' phosphate, demonstrating the importance of the downstream base in relaxing the Pol4 requirement. Combined with prior observations of Pol4-independent NHEJ of nicks with 5' hydroxyls, we suggest that base stacking interactions across the broken strands can stabilize a joint, allowing another polymerase to substitute for Pol4. This model predicts that a unique function of Pol4 is to actively stabilize template strands that lack stacking continuity. We also explored whether NHEJ end processing can occur via short- and long-patch pathways analogous to base excision repair. Results demonstrated that 5' dRPs could be removed in the absence of Pol4 lyase activity. The 5' flap endonuclease Rad27 was not required for repair in this or any situation tested, indicating that still other NHEJ 5' nucleases must exist.     

Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts

An extract from activated Xenopus eggs joins both matching and nonmatching ends of exogenous linear DNA substrates with high efficiency and fidelity (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907-924, 1988). In mammalian cells, such nonhomologous end joining (NHEJ) is known to require the Ku heterodimer, a component of DNA-dependent protein kinase. Here I investigated whether Ku is also required for the in vitro reaction in the egg extract. Immunological assays indicate that Ku is very abundant in the extract. I found that all NHEJ was inhibited by autoantibodies against Ku and that NHEJ between certain combinations of DNA ends was also decreased after immunodepletion of Ku from the extract. The formation of a joint between a DNA end with a 5'-protruding single strand (PSS) and an end with a 3'-PSS, between two ends with 3'-PSS, and between two blunt ends was most Ku dependent. On the other hand, NHEJ between two DNA ends bearing 5'-PSS was Ku independent. These results show that the Xenopus cell-free system will be useful to biochemically dissect the role of Ku in eukaryotic NHEJ.     

Non-homologous DNA end joining in the mature rat brain

Recent evidence suggests that DNA double strand breaks (DSBs) are introduced in neurons during the course of normal development, and that repair of such DSBs is essential for neuronal survival. Here we describe a non-homologous DNA end joining (NHEJ) system in the adult rat brain that may be used to repair DNA DSBs. In the brain NHEJ system, blunt DNA ends are joined with lower efficiency than cohesive or non-matching protruding ends. Moreover, brain NHEJ is blocked by DNA ligase inhibitors or by dATP and can occur in the presence or absence of exogenously added ATP. Comparison of NHEJ activities in several tissues showed that brain and testis share similar mechanisms for DNA end joining, whereas the activity in thymus seems to utilize different mechanisms than in the nervous system. The developmental profile of brain NHEJ showed increasing levels of activity after birth, peaking at postnatal day 12 and then gradually decreasing along with age. Brain distribution analysis in adult animals showed that NHEJ activity is differentially distributed among different regions. We suggest that the DNA NHEJ system may be utilized in the postnatal brain for the repair of DNA double strand breaks introduced within the genome in the postnatal brain.     

Mechanism of DNA double-strand break repair by non-homologous end joining

The repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Although the non-homologous end joining (NHEJ) pathway frequently results in minor changes in DNA sequence at the break site and occasionally the joining of previously unlinked DNA molecules, it is a major contributor to cell survival following exposure of mammalian cells to agents that cause DSBs. This repair mechanism is conserved in lower eukaryotes and in some prokaryotes although the majority of DSBs are repaired by recombinational repair pathways in these organisms. Here we will describe the biochemical properties of NHEJ factors from bacteria, Saccharomyces cerevisiae and mammals, and how physical and functional interactions among these factors co-ordinate the repair of DSBs.     

Repair of DNA double strand breaks by non-homologous end joining

DNA double strand breaks (DSB) are the most serious form of DNA damage. If not repaired they can lead to cell death. If misrepaired DSBs contribute to chromosomal aberrations and genomic instability. Non-homologous end joining (NHEJ) is one of two major pathways for the repair of DSBs in human cells. Proteins known to be required for NHEJ include the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis. This review discusses how these and other accessory proteins may function in the repair of DSBs produced by ionizing radiation (IR) and by V(D)J recombination.     

Mechanisms of overlap formation in nonhomologous DNA end joining.

Rejoining of nonhomologous DNA termini plays a central role in processes of illegitimate recombination. In Xenopus egg extracts, DNA ends with noncomplementary 4-nucleotide antiparallel single-strand protrusions are assumed to be joined by formation of short mismatched overlap intermediates. The extents of these overlaps may be set by single fortuitously matching base pairs and determine the patterns of subsequent gap filling and nick ligation. Under conditions of alternative overlap settings, rules for the most probable joining pathway and the effects of mismatches on junction formation were analyzed. We show that in certain cases, fill-in and ligation converting overlap intermediates into covalently closed junctions may proceed in the presence of unrepaired mismatches, whereas in other cases, completion of junction formation is preceded by removal of mismatches. Results are discussed in relation with "alignment" proteins postulated to structurally support overlap heteroduplexes during junction formation.     

A biochemically defined system for mammalian nonhomologous DNA end joining

Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.     

The role of DNA polymerase activity in human non-homologous end joining

In mammalian cells, DNA double-strand breaks are repaired mainly by non-homologous end joining, which modifies and ligates two DNA ends without requiring extensive base pairing interactions for alignment. We investigated the role of DNA polymerases in DNA-PK-dependent end joining of restriction-digested plasmids in vitro and in vivo. Rejoining of DNA blunt ends as well as those with partially complementary 5′ or 3′ overhangs was stimulated by 20–53% in HeLa cell-free extracts when dNTPs were included, indicating that part of the end joining is dependent on DNA synthesis. This DNA synthesis-dependent end joining was sensitive to aphidicolin, an inhibitor of α-like DNA polymerases. Furthermore, antibodies that neutralize the activity of DNA polymerase α were found to strongly inhibit end joining in vitro, whereas neutralizing antibodies directed against DNA polymerases β and did not. DNA sequence analysis of end joining products revealed two prominent modes of repair, one of which appeared to be dependent on DNA synthesis. Identical products of end joining were recovered from HeLa cells after transfection with one of the model substrates, suggesting that the same end joining mechanisms also operate in vivo. Fractionation of cell extracts to separate PCNA as well as depletion of cell extracts for PCNA resulted in a moderate but significant reduction in end joining activity, suggesting a potential role in a minor repair pathway.     

Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation.     

ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining

The human disorder ataxia telangiectasia (AT), which is characterized by genetic instability and neurodegeneration, results from mutation of the ataxia telangiectasia mutated (ATM) kinase. The loss of ATM leads to cell-cycle checkpoint deficiencies and other DNA damage signaling defects that do not fully explain all pathologies associated with A-T including neuronal loss. In addressing this enigma, we find here that ATM suppresses DNA double-strand break (DSB) repair by microhomology-mediated end joining (MMEJ). We show that ATM repression of DNA end-degradation is dependent on its kinase activities and that Mre11 is the major nuclease behind increased DNA end-degradation and MMEJ repair in A-T. Assessment of MMEJ by an in vivo reporter assay system reveals decreased levels of MMEJ repair in Mre11-knockdown cells and in cells treated with Mre11-nuclease inhibitor mirin. Structure-based modeling of Mre11 dimer engaging DNA ends suggests the 5' ends of a bridged DSB are juxtaposed such that DNA unwinding and 3'-5' exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5' termini and exonucleolytic degradation of the 3' ends in MMEJ. Together our results provide an integrated understanding of ATM and Mre11 in MMEJ: ATM has a critical regulatory function in controlling DNA end-stability and error-prone DSB repair and Mre11 nuclease plays a major role in initiating MMEJ in mammalian cells. These functions of ATM and Mre11 could be particularly important in neuronal cells, which are post-mitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs.     

Deficient DNA end joining activity in extracts from fanconi anemia fibroblasts

Fanconi anemia (FA) is a genetic disorder associated with genomic instability and cancer predisposition. Cultured cells from FA patients display a high level of spontaneous chromosome breaks and an increased frequency of intragenic deletions, suggesting that FA cells may have deficiencies in properly processing DNA double strand breaks. In this study, an in vitro plasmid DNA end joining assay was used to characterize the end joining capabilities of nuclear extracts from diploid FA fibroblasts from complementation groups A, C, and D. The Fanconi anemia extracts had 3-9-fold less DNA end joining activity and rejoined substrates with significantly less fidelity than normal extracts. Wild-type end joining activity could be reconstituted by mixing FA-D extracts with FA-A or FA-C extracts, while mixing FA-A and FA-C extracts had no effect on end joining activity. Protein expression levels of the DNA-dependent protein kinase (DNA-PK)/Ku-dependent nonhomologous DNA end-joining proteins Xrcc4, DNA ligase IV, Ku70, and Ku86 in FA and normal extracts were indistinguishable, as were DNA-dependent protein kinase and DNA end binding activities. The end joining activity as measured by the assay was not sensitive to the DNA-PK inhibitor wortmannin or dependent on the nonhomologous DNA end-joining factor Xrcc4. However, when DNA/protein ratios were lowered, the end joining activity became wortmannin-sensitive and no difference in end joining activity was observed between normal and FA extracts. Taken together, these results suggest that the FA fibroblast extracts have a deficiency in a DNA end joining process that is distinct from the DNA-PK/Ku-dependent nonhomologous DNA end joining pathway.     

Multiple barriers to nonhomologous DNA end joining during meiosis in Drosophila

Repair of meiotic double-strand breaks (DSBs) uses the homolog and recombination to yield crossovers while alternative pathways such as nonhomologous end joining (NHEJ) are suppressed. Our results indicate that NHEJ is blocked at two steps of DSB repair during meiotic prophase: first by the activity of the MCM-like protein MEI-218, which is required for crossover formation, and, second, by Rad51-related proteins SPN-B (XRCC3) and SPN-D (RAD51C), which physically interact and promote homologous recombination (HR). We further show that the MCM-like proteins also promote the activity of the DSB repair checkpoint pathway, indicating an early requirement for these proteins in DSB processing. We propose that when a meiotic DSB is formed in the absence of both MEI-218 and SPN-B or SPN-D, a DSB substrate is generated that can enter the NHEJ repair pathway. Indeed, due to its high error rate, multiple barriers may have evolved to prevent NHEJ activity during meiosis.