Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases λ and μ for nonhomologous end joining in human whole-cell extracts

XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5′ or 3′ overhangs, and no joining at all of partially complementary 3′ overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase λ, but was restored by addition of either polymerase λ or polymerase μ. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.     

Efficient processing of DNA ends during yeast nonhomologous end joining. Evidence for a DNA polymerase beta (Pol4)-dependent pathway.

Repair of DNA double strand breaks by nonhomologous end joining (NHEJ) requires enzymatic processing beyond simple ligation when the terminal bases are damaged or not fully compatible. We transformed yeast with a series of linearized plasmids to examine the role of Pol4 (Pol IV, DNA polymerase beta) in repair at a variety of end configurations. Mutation of POL4 did not impair DNA polymerase-independent religation of fully compatible ends and led to at most a 2-fold reduction in the frequency of joins that require only DNA polymerization. In contrast, the frequency of joins that also required removal of a 5'- or 3'-terminal mismatch was markedly reduced in pol4 (but not rev3, exo1, apn1, or rad1) yeast. In a chromosomal double strand break assay, pol4 mutation conferred a marked increase in sensitivity to HO endonuclease in a rad52 background, due primarily to loss of an NHEJ event that anneals with a 3'-terminal mismatch. The NHEJ activity of Pol4 was dependent on its nucleotidyl transferase function, as well as its unique amino terminus. Paradoxically, in vitro analyses with oligonucleotide substrates demonstrated that although Pol4 fills gaps with displacement of mismatched but not matched 5' termini, it lacks both 5'- and 3'-terminal nuclease activities. Pol4 is thus specifically recruited to perform gap-filling in an NHEJ pathway that must also involve as yet unidentified nucleases.     

A physical and functional interaction between yeast Pol4 and Dnl4-Lif1 links DNA synthesis and ligation in nonhomologous end joining

Genetic studies have implicated the Saccharomyces cerevisiae POL4 gene product in the repair of DNA double-strand breaks by nonhomologous end joining. Here we show that Pol4 preferentially catalyzes DNA synthesis on small gaps formed by the alignment of linear duplex DNA molecules with complementary ends, a DNA substrate specificity that is compatible with its predicted role in the repair of DNA double-strand breaks. Pol4 also interacts directly with the Dnl4 subunit of the Dnl4-Lif1 complex via its N-terminal BRCT domain. This interaction stimulates the DNA synthesis activity of Pol4 and, to a lesser extent, the DNA joining activity of Dnl4-Lif1. Notably, the joining of DNA substrates that require the combined action of Pol4 and Dnl4-Lif1 is much more efficient than the joining of similar DNA substrates that require only ligation. Thus, the physical and functional interactions between Pol4 and Dnl4-Lif1 provide a molecular mechanism for both the recruitment of Pol4 to in vivo DNA double-strand breaks and the coupling of the gap filling DNA synthesis and DNA joining reactions that complete the microhomology-mediated pathway of nonhomologous end joining.     

Essential factors for incompatible DNA end joining at chromosomal DNA double strand breaks in vivo

Non-homologous end joining (NHEJ) is a major pathway for the repair of DNA double strand break (DSBs) with incompatible DNA ends, which are often generated by ionizing irradiation. In vitro reconstitution studies have indicated that NHEJ of incompatible DNA ends requires not only the core steps of synapsis and ligation, employing KU80/DNA-PKcs and LIG4, but also additional DNA end processing steps, such as DNA end resection by Artemis and gap-filling by POLλ and POLμ. It seems that DNA end processing steps are important for joining of incompatible DNA ends rather than compatible ends. Despite the fact that DNA end processing is important for incompatible DNA end joining in vitro, the role of DNA processing in NHEJ of incompatible DSBs in vivo has not yet been demonstrated. Here we investigated the in vivo roles of proteins implicated in each step of NHEJ using an assay in which NHEJ of incompatible DNA ends on chromosomal DNA can be assessed in living human cells. siRNA- or inhibitor-mediated impairment of factors in each NHEJ step resulted in a reduction in joining efficiency. Strikingly, stronger effects were observed when DNA end resection and ligation protein functions were impaired. Disruption of synapsis by KU80 and DNA-PKcs impairment, or the disruption of gap filling by POLλ and POLμ depletion, resulted in higher levels of microhomology-mediated joining. The present study indicates that DNA end resection and ligation factors are critical for the efficient joining of incompatible ends in vivo, further emphasizing the importance of synapsis and gap-filling factors in preventing illegitimate joining.     

DNA joint dependence of pol X family polymerase action in nonhomologous end joining

DNA double strand breaks (DSBs) can be rejoined directly by the nonhomologous end-joining (NHEJ) pathway of repair. Nucleases and polymerases are required to promote accurate NHEJ when the terminal bases of the DSB are damaged. The same enzymes also participate in imprecise rejoining and joining of incompatible ends, important mutagenic events. Previous work has shown that the Pol X family polymerase Pol4 is required for some but not all NHEJ events that require gap filling in Saccharomyces cerevisiae. Here, we systematically analyzed DSB end configurations and found that gaps on both strands and overhang polarity are the principal factors that determine whether a joint requires Pol4. DSBs with 3'-overhangs and a gap on each strand strongly depended on Pol4 for repair, DSBs with 5'-overhangs of the same sequence did not. Pol4 was not required when 3'-overhangs contained a gap on only one strand, however. Pol4 was equally required at 3'-overhangs of all lengths within the NHEJ-dependent range but was dispensable outside of this range, indicating that Pol4 is specific to NHEJ. Loss of Pol4 did not affect the rejoining of DSBs that utilized a recessed microhomology or DSBs bearing 5'-hydroxyls but no gap. Finally, mammalian Pol X polymerases were able to differentially complement a pol4 mutation depending on the joint structure, demonstrating that these polymerases can participate in yeast NHEJ but with distinct properties.     

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.     

Implication of DNA polymerase lambda in alignment-based gap filling for nonhomologous DNA end joining in human nuclear extracts

Accurate repair of free radical-mediated DNA double-strand breaks by the nonhomologous end joining pathway requires replacement of fragmented nucleotides in the aligned ends by a gap-filling DNA polymerase. Nuclear extracts of human HeLa cells, supplemented with recombinant XRCC4-DNA ligase IV complex (XRCC4/ligase IV), were capable of accurately rejoining model double-strand break substrates with a 1- or 2-base gap, and the gap-filling step was dependent on XRCC4/ligase IV. To determine what polymerase was responsible for gap filling, end joining was examined in the presence of polyclonal antibodies against each of two prime candidate enzymes, DNA polymerases mu and lambda, both of which were present in the extracts. For a DNA substrate with partially complementary 3' overhangs and a 2-base gap, antibodies to polymerase lambda completely eliminated both gap filling and accurate end joining, whereas antibodies to polymerase mu had little effect. Immunodepletion of polymerase lambda, but not polymerase mu, likewise blocked both gap filling and end joining, and both functions could be restored by addition of recombinant polymerase lambda. Recombinant polymerase mu, and a truncated polymerase lambda lacking the Brca1 C-terminal domain, were at least 10-fold less active in restoring gap filling to the immunodepleted extracts, and polymerase beta was completely inactive. The results suggest that polymerase lambda is the primary gap-filling polymerase for accurate nonhomologous end joining, and that the Brca1 C-terminal domain is required for this activity.     

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.     

Mismatch tolerance by DNA polymerase Pol4 in the course of nonhomologous end joining in Saccharomyces cerevisiae

In yeast, the nonhomologous end joining pathway (NHEJ) mobilizes the DNA polymerase Pol4 to repair DNA double-strand breaks when gap filling is required prior to ligation. Using telomere-telomere fusions caused by loss of the telomeric protein Rap1 and double-strand break repair on transformed DNA as assays for NHEJ between fully uncohesive ends, we show that Pol4 is able to extend a 3'-end whose last bases are mismatched, i.e., mispaired or unpaired, to the template strand.     

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.     

Tolerance for 8-oxoguanine but not thymine glycol in alignment-based gap filling of partially complementary double-strand break ends by DNA polymerase lambda in human nuclear extracts

Ionizing radiation induces various clustered DNA lesions, including double-strand breaks (DSBs) accompanied by nearby oxidative base damage. Previous work showed that, in HeLa nuclear extracts, DSBs with partially complementary 3′ overhangs and a one-base gap in each strand are accurately rejoined, with the gaps being filled by DNA polymerase λ. To determine the possible effect of oxidative base damage on this process, plasmid substrates were constructed containing overhangs with 8-oxoguanine or thymine glycol in base-pairing positions of 3-base (-ACG or -GTA) 3′ overhangs. In this context, 8-oxoguanine was well tolerated by the end-joining machinery when present at one end of the break, but not when present at both ends. Thymine glycol was less well tolerated than 8-oxoguanine, reducing gap filling and accurate rejoining by at least 10-fold. The results suggest that complex DSBs can be accurately rejoined despite the presence of accompanying base damage, but that nonplanar bases constitute a major barrier to this process and promote error-prone joining. A chimeric DNA polymerase, in which the catalytic domain of polymerase λ was replaced with that of polymerase β, could not substitute for polymerase λ in these assays, suggesting that this domain is specifically adapted for gap filling on aligned DSB ends.     

A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining

Three Pol X family members have been linked to nonhomologous end joining (NHEJ) in mammals. Template-independent TdT promotes diversity during NHEJ-dependent repair of V(D)J recombination intermediates, but the roles of the template-dependent polymerases mu and lambda in NHEJ remain unclear. We show here that pol mu and pol lambda are similarly recruited by NHEJ factors to fill gaps when ends have partially complementary overhangs, suggesting equivalent roles promoting accuracy in NHEJ. However, only pol mu promotes accuracy during immunoglobulin kappa recombination. This distinctive in vivo role correlates with the TdT-like ability of pol mu, but not pol lambda, to act when primer termini lack complementary bases in the template strand. However, unlike TdT, synthesis by pol mu in this context is primarily instructed by a template from another DNA molecule. This apparent gradient of template dependence is largely attributable to a small structural element that is present but different in all three polymerases.     

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.     

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.     

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.     

End-bridging is required for pol mu to efficiently promote repair of noncomplementary ends by nonhomologous end joining

DNA polymerase μ is a member of the mammalian pol X family and reduces deletion during chromosome break repair by nonhomologous end joining (NHEJ). This biological role is linked to pol μ's ability to promote NHEJ of ends with noncomplementary 3′ overhangs, but questions remain regarding how it performs this role. We show here that synthesis by pol μ in this context is often rapid and, despite the absence of primer/template base-pairing, instructed by template. However, pol μ is both much less active and more prone to possible template independence in some contexts, including ends with overhangs longer than two nucleotides. Reduced activity on longer overhangs implies pol μ is less able to synthesize across longer gaps, arguing pol μ must bridge both sides of gaps between noncomplementary ends to be effective in NHEJ. Consistent with this argument, a pol μ mutant defective specifically on gapped substrates is also less active during NHEJ of noncomplementary ends both in vitro and in cells. Taken together, pol μ activity during NHEJ of noncomplementary ends can thus be primarily linked to pol μ's ability to work together with core NHEJ factors to bridge DNA ends and perform a template-dependent gap fill-in reaction.     

Polynucleotide kinase and aprataxin-like forkhead-associated protein (PALF) acts as both a single-stranded DNA endonuclease and a single-stranded DNA 3' exonuclease and can participate in DNA end joining in a biochemical system

Polynucleotide kinase and aprataxin-like forkhead-associated protein (PALF, also called aprataxin- and PNK-like factor (APLF)) has been shown to have nuclease activity and to use its forkhead-associated domain to bind to x-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4). Because XRCC4 is a key component of the ligase IV complex that is central to the nonhomologous DNA end joining (NHEJ) pathway, this raises the possibility that PALF might play a role in NHEJ. For this reason, we further studied the nucleolytic properties of PALF, and we searched for any modulation of PALF by NHEJ components. We verified that PALF has 3' exonuclease activity. However, PALF also possesses single-stranded DNA endonuclease activity. This single-stranded DNA endonuclease activity can act at all single-stranded sites except those within four nucleotides 3' of a double-stranded DNA junction, suggesting that PALF minimally requires approximately four nucleotides of single-strandedness. Ku, DNA-dependent protein kinase catalytic subunit, and XRCC4-DNA ligase IV do not modulate PALF nuclease activity on single-stranded DNA or overhangs of duplex substrates. PALF does not open DNA hairpins. However, in a reconstituted end joining assay that includes Ku, XRCC4-DNA ligase IV, and PALF, PALF is able to resect 3' overhanging nucleotides and permit XRCC4-DNA ligase IV to complete the joining process in a manner that is as efficient as Artemis. Reduction of PALF in vivo reduces the joining of incompatible DNA ends. Hence, PALF can function in concert with other NHEJ proteins.     

XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps

XRCC4 and DNA ligase IV form a complex that is essential for the repair of all double-strand DNA breaks by the nonhomologous DNA end joining pathway in eukaryotes. We find here that human XRCC4:DNA ligase IV can ligate two double-strand DNA ends that have fully incompatible short 3' overhang configurations with no potential for base pairing. Moreover, at DNA ends that share 1-4 annealed base pairs, XRCC4:DNA ligase IV can ligate across gaps of 1 nt. Ku can stimulate the joining, but is not essential when there is some terminal annealing. Polymerase mu can add nucleotides in a template-independent manner under physiological conditions; and the subset of ends that thereby gain some terminal microhomology can then be ligated. Hence, annealing at sites of microhomology is very important, but the flexibility of the ligase complex is paramount in nonhomologous DNA end joining. These observations provide an explanation for several in vivo observations that were difficult to understand previously.     

End-joining repair of double-strand breaks in Drosophila melanogaster is largely DNA ligase IV independent

Repair of DNA double-strand breaks can occur by either nonhomologous end joining or homologous recombination. Most nonhomologous end joining requires a specialized ligase, DNA ligase IV (Lig4). In Drosophila melanogaster, double-strand breaks created by excision of a P element are usually repaired by a homologous recombination pathway called synthesis-dependent strand annealing (SDSA). SDSA requires strand invasion mediated by DmRad51, the product of the spn-A gene. In spn-A mutants, repair proceeds through a nonconservative pathway involving the annealing of microhomologies found within the 17-nt overhangs produced by P excision. We report here that end joining of P-element breaks in the absence of DmRad51 does not require Drosophila LIG4. In wild-type flies, SDSA is sometimes incomplete, and repair is finished by an end-joining pathway that also appears to be independent of LIG4. Loss of LIG4 does not increase sensitivity to ionizing radiation in late-stage larvae, but lig4 spn-A double mutants do show heightened sensitivity relative to spn-A single mutants. Together, our results suggest that a LIG4-independent end-joining pathway is responsible for the majority of double-strand break repair in the absence of homologous recombination in flies.     

Trimming of damaged 3′ overhangs of DNA double-strand breaks by the Metnase and Artemis endonucleases

Both Metnase and Artemis possess endonuclease activities that trim 3′ overhangs of duplex DNA. To assess the potential of these enzymes for facilitating resolution of damaged ends during double-strand break rejoining, substrates bearing a variety of normal and structurally modified 3′ overhangs were constructed, and treated either with Metnase or with Artemis plus DNA-dependent protein kinase (DNA-PK). Unlike Artemis, which trims long overhangs to 4–5 bases, cleavage by Metnase was more evenly distributed over the length of the overhang, but with significant sequence dependence. In many substrates, Metnase also induced marked cleavage in the double-stranded region within a few bases of the overhang. Like Artemis, Metnase efficiently trimmed overhangs terminated in 3′-phosphoglycolates (PGs), and in some cases the presence of 3′-PG stimulated cleavage and altered its specificity. The nonplanar base thymine glycol in a 3′ overhang severely inhibited cleavage by Metnase in the vicinity of the modified base, while Artemis was less affected. Nevertheless, thymine glycol moieties could be removed by Metnase- or Artemis-mediated cleavage at sites farther from the terminus than the lesion itself. In in vitro end-joining systems based on human cell extracts, addition of Artemis, but not Metnase, effected robust trimming of an unligatable 3′-PG overhang, resulting in a dramatic stimulation of ligase IV- and XLF-dependent end joining. Thus, while both Metnase and Artemis are biochemically capable of resolving a variety of damaged DNA ends for the repair of complex double-strand breaks, Artemis appears to act more efficiently in the context of other nonhomologous end joining proteins.