Structure and function of a mycobacterial NHEJ DNA repair polymerase

Non homologous end-joining (NHEJ)-mediated repair of DNA double-strand breaks in prokaryotes requires Ku and a specific multidomain DNA ligase (LigD). We present crystal structures of the primase/polymerisation domain (PolDom) of Mycobacterium tuberculosis LigD, alone and complexed with nucleotides. The PolDom structure combines the general fold of the archaeo-eukaryotic primase (AEP) superfamily with additional loops and domains that together form a deep cleft on the surface, likely used for DNA binding. Enzymatic analysis indicates that the PolDom of LigD, even in the absence of accessory domains and Ku proteins, has the potential to recognise DNA end-joining intermediates. Strikingly, one of the main signals for the specific and efficient binding of PolDom to DNA is the presence of a 5'-phosphate group, located at the single/double-stranded junction at both gapped and 3'-protruding DNA molecules. Although structurally unrelated, Pol lambda and Pol mu, the two eukaryotic DNA polymerases involved in NHEJ, are endowed with a similar capacity to bind a 5'-phosphate group. Other properties that are beneficial for NHEJ, such as the ability to generate template distortions and realignments of the primer, displayed by Pol lambda and Pol mu, are shared by the PolDom of bacterial LigD. In addition, PolDom can perform non-mutagenic translesion synthesis on termini containing modified bases. Significantly, ribonucleotide insertion appears to be a recurrent theme associated with NHEJ, maximised in this case by the deployment of a dedicated primase, although its in vivo relevance is unknown.     

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.     

Repair of Double-Strand DNA Breaks by the Human Nonhomologous DNA End Joining Pathway: The Iterative Processing Model

Naturally-occurring ionizing radiation and reactive oxygen species (ROS) from oxidative metabolism are factors that have challenged all life forms during the course of evolution. Ionizing radiation (IR) and reactive oxygen species cause a diverse set of double-strand DNA end configurations. Nonhomologous DNA end joining (NHEJ) is an optimal DNA repair pathway for dealing with such a diverse set of DNA lesions. NHEJ can carry out nucleolytic, polymerase, and ligation operations on each strand independently. This iterative processing nature of NHEJ is ideal for repair of pathologic and physiologic double-strand breaks because it permits sequential action of the NHEJ enzymes on each DNA end and on each strand. The versatility of the Artemis:DNA-PKcs endonuclease in cleaving 5’ and 3’ overhangs, hairpins, gaps, flaps, and various loop conformations makes it well-suited for DNA end modifications on oxidized overhangs. In addition, the ability to cleave stem-loop and hairpin structures permits it to open terminal fold-back configurations that may arise at DNA ends after IR damage. The ability of the XRCC4:DNA ligase IV complex to ligate one strand without ligation of the other permits additional end joining flexibility in NHEJ and raises the possibility of optional involvement of repair proteins from other pathways.     

Ribonucleotides and manganese ions improve non-homologous end joining by human Polμ

Human DNA polymerase mu (Polμ), a family X member involved in DNA repair, has both template-directed and terminal transferase (template-independent) activities. In addition to their ability to incorporate untemplated nucleotides, another similarity between Polµ and terminal deoxynucleotidyl transferase (TdT) is their promiscuity in using ribonucleotides (NTPs), whose physiological significance is presently unknown. As shown here, Polµ can use NTPs instead of deoxynucleotides (dNTPs) during non-homologous end joining (NHEJ) of non-complementary ends, a Polµ-specific task. Moreover, a physiological concentration of Mn²⁺ ions did benefit Polµ-mediated NHEJ by improving the efficiency and accuracy of nucleotide insertion. Analysis of different mutations in the ‘steric gate’ of the active site indicated that Polµ is taking advantage of an open active site, valid for selecting alternative activating metal ions and nucleotides as substrates. This versatility would allow ad hoc selection of the most appropriate nucleotide/metal ion combination for individual NHEJ events to gain efficiency without a cost in terms of fidelity, thus widening the spectrum of available solutions to position a discontinuous template strand in proper register for connection.     

DNA polymerase lambda can elongate on DNA substrates mimicking non-homologous end joining and interact with XRCC4-ligase IV complex

Non-homologous end joining (NHEJ) is one of two pathways responsible for the repair of double-strand breaks in eukaryotic cells. The mechanism involves the alignment of broken DNA ends with minimal homology, fill in of short gaps by DNA polymerase(s), and ligation by XRCC4-DNA ligase IV complex. The gap-filling polymerase has not yet been positively identified, but recent biochemical studies have implicated DNA polymerase lambda (pol lambda), a novel DNA polymerase that has been assigned to the pol X family, in this process. Here we demonstrate that purified pol lambda can efficiently catalyze gap-filling synthesis on DNA substrates mimicking NHEJ. By designing two truncated forms of pol lambda, we also show that the unique proline-rich region in pol lambda plays a role in limiting strand displacement synthesis, a feature that may help its participation in in vivo NHEJ. Moreover, pol lambda interacts with XRCC4-DNA ligase IV via its N-terminal BRCT domain and the interaction stimulates the DNA synthesis activity of pol lambda. Taken together, these data strongly support that pol lambda functions in DNA polymerization events during NHEJ.     

Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency

The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PK_cs and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5′ overhangs, and 3′ overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.     

Insights into the replisome from the structure of a ternary complex of the DNA polymerase III alpha-subunit

The crystal structure of the catalytic α−subunit of the DNA polymerase III (PolIIIα) holoenzyme bound to primer-template DNA and an incoming deoxy-nucleoside 5′-triphosphate has been determined at 4.6-Å resolution. The polymerase interacts with the sugar-phosphate backbone of the DNA across its minor groove, which is made possible by significant movements of the thumb, finger, and β-binding domains relative to their orientations in the unliganded polymerase structure. Additionally, the DNA and incoming nucleotide are bound to the active site of PolIIIα nearly identically as they are in their complex with DNA polymerase β, thereby proving that the eubacterial replicating polymerase, but not the eukaryotic replicating polymerase, is homologous to DNA polymerase β. Finally, superimposing a recent structure of the clamp bound to DNA on this PolIIIα complex with DNA places a loop of the β-binding domain into the appropriate clamp cleft and supports a mechanism of polymerase switching.     

Gap Filling Activities of Pseudomonas DNA Ligase D (LigD) Polymerase and Functional Interactions of LigD with the DNA End-binding Ku Protein

Many bacterial pathogens, including Pseudomonas aeruginosa, have a nonhomologous end joining (NHEJ) system of DNA double strand break (DSB) repair driven by Ku and DNA ligase D (LigD). LigD is a multifunctional enzyme composed of a ligase domain fused to an autonomous polymerase module (POL) that adds ribonucleotides or deoxyribonucleotides to DSB ends and primer-templates. LigD POL and the eukaryal NHEJ polymerase λ are thought to bridge broken DNA ends via contacts with a duplex DNA segment downstream of the primer terminus, a scenario analogous to gap repair. Here, we characterized the gap repair activity of Pseudomonas LigD POL, which is more efficient than simple templated primer extension and relies on a 5′-phosphate group on the distal gap strand end to confer apparent processivity in filling gaps of 3 or 4 nucleotides. Mutations of the His-553, Arg-556, and Lys-566 side chains implicated in DNA 5′-phosphate binding eliminate the preferential filling of 5′-phosphate gaps. Mutating Phe-603, which is imputed to stack on the nucleobase of the template strand that includes the 1st bp of the downstream gap duplex segment, selectively affects incorporation of the final gap-closing nucleotide. We find that Pseudomonas Ku stimulates POL-catalyzed ribonucleotide addition to a plasmid DSB end and promotes plasmid end joining by full-length Pseudomonas LigD. A series of incremental truncations from the C terminus of the 293-amino acid Ku polypeptide identifies Ku-(1–229) as sufficient for homodimerization and LigD stimulation. The slightly longer Ku-(1–253) homodimer forms stable complexes at both ends of linear plasmid DNA that protect the DSBs from digestion by 5′- and 3′-exonucleases.     

The Mre11/Rad50/Xrs2 complex and non-homologous end-joining of incompatible ends in S. cerevisiae

In Saccharomyces cerevisiae, the Mre11/Rad50/Xrs2 (MRX) complex plays important roles in both homologous and non-homologous pathways of DNA repair. In this study, we investigated the role of the MRX complex and its enzymatic functions in non-homologous repair of DNA ends containing incompatible end structures. Using a plasmid transformation assay, we found that mre11 and rad50 null strains are extremely deficient in joining of incompatible DNA ends. Expression of the nuclease-deficient Mre11 mutant H125N fully complemented the mre11 strain for joining of mismatched ends in the absence of homology, while a mutant of Rad50 deficient in ATP-dependent activities exhibited levels of end-joining similar to a rad50 deletion strain. Although the majority of non-homologous end-joining (NHEJ) products isolated did not contain microhomologies, introduction of an 8bp microhomology at mismatched ends resulted in microhomology-mediated joining in all of the products recovered, demonstrating that a microhomology exerts a dominant effect on processing events that occur during NHEJ. Nuclease-deficient Mre11p was less efficient in promoting microhomology-mediated end-joining in comparison to its ability to stimulate non-microhomology-mediated events, suggesting that Mre11p influences, but is not essential for, microhomology-mediated repair. When the linearized DNA was transformed in the presence of an intact homologous plasmid to facilitate gap repair, there was no decrease in NHEJ products obtained, suggesting that NHEJ and homologous repair do not compete for DNA ends in vivo. These results suggest that the MRX complex is essential for joining of incompatible ends by NHEJ, and the ATP-dependent activities of Rad50 are critical for this process.     

A structural solution for the DNA polymerase lambda-dependent repair of DNA gaps with minimal homology

Human DNA polymerase lambda (Pol lambda) is a family X member with low frameshift fidelity that has been suggested to perform gap-filling DNA synthesis during base excision repair and during repair of broken ends with limited homology. Here, we present a 2.1 A crystal structure of the catalytic core of Pol lambda in complex with DNA containing a two nucleotide gap. Pol lambda makes limited contacts with the template strand at the polymerase active site, and superimposition with Pol beta in a ternary complex suggests a shift in the position of the DNA at the active site that is reminiscent of a deletion intermediate. Surprisingly, Pol lambda can adopt a closed conformation, even in the absence of dNTP binding. These observations have implications for the catalytic mechanism and putative DNA repair functions of Pol lambda.     

Nonhomologous end-joining in a cell-free extract from the cultured silkworm cell line BmN4

Nonhomologous end-joining (NHEJ) is one of the repair pathways for double-strand breaks (DSBs) in eukaryotic cells. By using linearized plasmid substrates, we have detected intramolecular NHEJ activity in a cell-free extract from the cultured silkworm cell line BmN4. The efficiency of NHEJ differed according to the structure of DNA ends; approximately 1% of input DNA was repaired when the substrate had cohesive ends. The reaction required the hydrolysis of nucleotide triphosphate; interestingly, all of four rNTPs or four dNTPs could support the reaction. A substrate with non-complementary DNA ends was mainly repaired by the DNA polymerase-mediated pathway. These results indicate that the present cell-free system will be useful to analyze the molecular mechanisms of DSB repair and NHEJ in insect cells.     

Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis

We report the crystal structures of a translesion DNA polymerase, Dpo4, complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. These structures suggest two mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. By replacing Mg2+ with Mn2+, which has a relaxed coordination requirement and tolerates misalignment, the catalytic efficiency of misincorporation increases dramatically. Mn2+ also enhances translesion synthesis by Dpo4. Subtle conformational changes that lead to the proper metal ion coordination may, therefore, be a key step in catalysis. Second, the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and degrades the primer strand in the presence of pyrophosphate. The correct incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an incorrect incoming nucleotide does not.     

End-processing nucleases and phosphodiesterases: An elite supporting cast for the non-homologous end joining pathway of DNA double-strand break repair

Nonhomologous end joining (NHEJ) is an error-prone DNA double-strand break repair pathway that is active throughout the cell cycle. A substantial fraction of NHEJ repair events show deletions and, less often, insertions in the repair joints, suggesting an end-processing step comprising the removal of mismatched or damaged nucleotides by nucleases and other phosphodiesterases, as well as subsequent strand extension by polymerases. A wide range of nucleases, including Artemis, Metnase, APLF, Mre11, CtIP, APE1, APE2 and WRN, are biochemically competent to carry out such double-strand break end processing, and have been implicated in NHEJ by at least circumstantial evidence. Several additional DNA end-specific phosphodiesterases, including TDP1, TDP2 and aprataxin are available to resolve various non-nucleotide moieties at DSB ends. This review summarizes the biochemical specificities of these enzymes and the evidence for their participation in the NHEJ pathway.     

Single-stranded DNA ligation and XLF-stimulated incompatible DNA end ligation by the XRCC4-DNA ligase IV complex: influence of terminal DNA sequence

The double-strand DNA break repair pathway, non-homologous DNA end joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and ligase activities. Here we find that the joining of ends by XRCC4-ligase IV is markedly influenced by the terminal sequence, and a steric hindrance model can account for this. XLF (Cernunnos) stimulates the joining of both incompatible DNA ends and compatible DNA ends at physiologic concentrations of Mg²⁺, but only of incompatible DNA ends at higher concentrations of Mg²⁺, suggesting charge neutralization between the two DNA ends within the ligase complex. XRCC4-DNA ligase IV has the distinctive ability to ligate poly-dT single-stranded DNA and long dT overhangs in a Ku- and XLF-independent manner, but not other homopolymeric DNA. The dT preference of the ligase is interesting given the sequence bias of the NHEJ polymerase. These distinctive properties of the XRCC4-DNA ligase IV complex explain important aspects of its in vivo roles.     

End-processing during non-homologous end-joining: a role for exonuclease 1

Non-homologous end-joining (NHEJ) is a critical error-prone pathway of double strand break repair. We recently showed that tyrosyl DNA phosphodiesterase 1 (Tdp1) regulates the accuracy of NHEJ repair junction formation in yeast. We assessed the role of other enzymes in the accuracy of junction formation using a plasmid repair assay. We found that exonuclease 1 (Exo1) is important in assuring accurate junction formation during NHEJ. Like tdp1Δ mutants, exo1Δ yeast cells repairing plasmids with 5'-extensions can produce repair junctions with templated insertions. We also found that exo1Δ mutants have a reduced median size of deletions when joining DNA with blunt ends. Surprisingly, exo1Δ pol4Δ mutants repair blunt ends with a very low frequency of deletions. This result suggests that there are multiple pathways that process blunt ends prior to end-joining. We propose that Exo1 acts at a late stage in end-processing during NHEJ. Exo1 can reverse nucleotide additions occurring due to polymerization, and may also be important for processing ends to expose microhomologies needed for NHEJ. We propose that accurate joining is controlled at two steps, a first step that blocks modification of DNA ends, which requires Tdp1, and a second step that occurs after synapsis that requires Exo1.     

A RAP1/TRF2 complex inhibits nonhomologous end-joining at human telomeric DNA ends

The mechanisms by which telomeres are distinguished from DNA double-strand breaks are poorly understood. Here we have defined the minimal requirements for the protection of telomeric DNA ends from nonhomologous end-joining (NHEJ). Neither long, single-stranded overhangs nor t loop formation is essential to prevent NHEJ-mediated ligation of telomeric ends in vitro. Instead, a tandem array of 12 telomeric repeats is sufficient to impede illegitimate repair in a highly directional manner at nearby DNA ends. The polarity of end protection is consistent with the orientation of naturally occurring telomeres and is well suited to minimize interference between chromosome capping and the repair of DNA double-strand breaks in subtelomeric sequences. Biochemical fractionation and reconstitution revealed that telomere protection is mediated by a RAP1/TRF2 complex, providing evidence for a direct role for human RAP1 in the protection of telomeric DNA from NHEJ.     

Lesion bypass of N2-ethylguanine by human DNA polymerase iota

Nucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase iota was measured and structures of the DNA polymerase iota-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase iota opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase iota incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase iota opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase iota extends from the N2-ethyl-Gua:Cyt 3' terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase iota catalyzed reaction is well suited for N(2)-ethyl-Gua bypass. The structure of DNA polymerase iota with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase iota complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase iota polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase iota efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.     

Proofreading activity of DNA polymerase Pol2 mediates 3'-end processing during nonhomologous end joining in yeast

Genotoxic agents that cause double-strand breaks (DSBs) often generate damage at the break termini. Processing enzymes, including nucleases and polymerases, must remove damaged bases and/or add new bases before completion of repair. Artemis is a nuclease involved in mammalian nonhomologous end joining (NHEJ), but in Saccharomyces cerevisiae the nucleases and polymerases involved in NHEJ pathways are poorly understood. Only Pol4 has been shown to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends. We previously developed a chromosomal DSB assay in yeast to study factors involved in NHEJ. Here, we use this system to examine DNA polymerases required for NHEJ in yeast. We demonstrate that Pol2 is another major DNA polymerase involved in imprecise end joining. Pol1 modulates both imprecise end joining and more complex chromosomal rearrangements, and Pol3 is primarily involved in NHEJ-mediated chromosomal rearrangements. While Pol4 is the major polymerase to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends, Pol2 is important for the recession of 3' flaps that can form during imprecise pairing. Indeed, a mutation in the 3'-5' exonuclease domain of Pol2 dramatically reduces the frequency of end joins formed with initial 3' flaps. Thus, Pol2 performs a key 3' end-processing step in NHEJ.     

The Mre11/Rad50/Nbs1 complex functions in resection-based DNA end joining in Xenopus laevis

The repair of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. In higher eukaryotes, DNA DSBs are predominantly repaired by non-homologous end joining (NHEJ), but DNA ends can also be joined by an alternative error-prone mechanism termed microhomology-mediated end joining (MMEJ). In MMEJ, the repair of DNA breaks is mediated by annealing at regions of microhomology and is always associated with deletions at the break site. In budding yeast, the Mre11/Rad5/Xrs2 complex has been demonstrated to play a role in both classical NHEJ and MMEJ, but the involvement of the analogous MRE11/RAD50/NBS1 (MRN) complex in end joining in higher eukaryotes is less certain. Here we demonstrate that in Xenopus laevis egg extracts, the MRN complex is not required for classical DNA-PK-dependent NHEJ. However, the XMRN complex is necessary for resection-based end joining of mismatched DNA ends. This XMRN-dependent end joining process is independent of the core NHEJ components Ku70 and DNA-PK, occurs with delayed kinetics relative to classical NHEJ and brings about repair at sites of microhomology. These data indicate a role for the X. laevis MRN complex in MMEJ.     

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.