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Mycobacterial Ku and Ligase Proteins Constitute a Two-Component NHEJ Repair Machine

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Science  22 Oct 2004:
Vol. 306, Issue 5696, pp. 683-685
DOI: 10.1126/science.1099824

Abstract

In mammalian cells, repair of DNA double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ) is critical for genome stability. Although the end-bridging and ligation steps of NHEJ have been reconstituted in vitro, little is known about the end-processing reactions that occur before ligation. Recently, functionally homologous end-bridging and ligation activities have been identified in prokarya. Consistent with its homology to polymerases and nucleases, we demonstrate that DNA ligase D from Mycobacterium tuberculosis (Mt-Lig) possesses a unique variety of nucleotidyl transferase activities, including gap-filling polymerase, terminal transferase, and primase, and is also a 3′ to 5′ exonuclease. These enzyme activities allow the Mt-Ku and Mt-Lig proteins to join incompatible DSB ends in vitro, as well as to reconstitute NHEJ in vivo in yeast. These results demonstrate that prokaryotic Ku and ligase form a bona fide NHEJ system that encodes all the recognition, processing, and ligation activities required for DSB repair.

Nonhomologous end-joining (NHEJ) is the major pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells (1). Key factors in eukaryotic NHEJ are the Ku70/Ku80 heterodimer (Ku) and DNA ligase IV (2), which have functional homologs in prokaryotes (3). Ku binds directly to the termini of DSBs and has end-bridging activity (4, 5), as has the yeast Mre11/Rad50/Xrs2 (MRX) complex (6). These factors interact with ligase IV/XRCC4 to achieve repair (68). Many DSBs generated in vivo have damaged or non-complementary termini that require processing by nucleases and polymerases to generate ligatable termini. The molecular mechanisms of these reactions are poorly understood.

The Mycobacterium tuberculosis DNA repair ligase, Mt-Lig (Rv0938 and LigD), which is specifically stimulated by Mt-Ku homodimer (3), contains domains that exhibit significant homology with polymerases (912) and possibly nucleases (1012), suggesting that Mt-Lig might process and then join incompatible DNA ends. Mt-Lig is most homologous to eukaryotic primase (912); however, a requirement for primase during NHEJ is not obvious. Eukaryotic primases share significant sequence homology with the Pol X family of nucleotidyl transferases active in NHEJ (1317). Purified recombinant Mt-Lig (3, 18) was an efficient DNA-dependent DNA polymerase in template-dependent primer extension assays (fig. S1). Mt-Lig similarly acted as a DNA-dependent RNA polymerase (fig. S1). Mutating predicted catalytic aspartate residues abolished all polymerase activities, confirming that they are intrinsic properties of Mt-Lig (fig. S1). Terminal transferase (TdT), a Pol X family member, has also been implicated in V(D)J recombination and NHEJ (14, 15). Mt-Lig used adenosine triphosphate (ATP) and, to a lesser extent, dATP to extend single-strand (ss) DNA, demonstrating terminal transferase activity (fig. S2). Finally, Mt-Lig synthesized 10- to 25–nucleotide (nt) oligomers on unprimed circular M13 ssDNA using nucleotide triphosphates (NTPs) but not dNTPs (fig. S3). Thus, Mt-Lig has DNA-dependent RNA primase activity.

To examine microhomology-mediated joints typical of NHEJ, we performed polymerization assays with oligonucleotides that generate a nonligatable 1-nt gap upon alignment (Fig. 1A). Mt-Lig efficiently filled in the gap with no detectable strand displacement synthesis (Fig. 1A, left). Addition of a phosphate group to the 5′ terminus of the 1-nt gap resulted in gap filling and ligation (Fig. 1A, right), indicating the concerted action of Mt-Lig polymerase and ligase activities.

Fig. 1.

Mt-Ligase is a multidomain enzyme with polymerase, nuclease, and ligase activities. (A) Left: In assays with DNA duplexes that form a non-ligatable 1-nt gap, Mt-Lig (0.125, 0.25, 0.5, or 1 μM) efficiently filled in the gap. Right: When a phosphate group was added to the 5′ terminus, Mt-Lig (1 μM) ligated the filled intermediate. (B) DNA duplexes with 3′-overhangs were incubated with increasing amounts of Mt-Lig (0.25, 0.5, and 1 μM). The positions of the 51-nt oligomer substrate and 43-nt oligomer degradation products are indicated. (C) DNA substrates were incubated with increasing amounts of Mt-Lig (0.25, 0.5, and 1 μM). The positions of the 51-nt oligomer substrate and 48- and 43-nt oligomer 3′ degradation products are indicated.

Mt-Lig is also predicted to contain a distinct nuclease domain (10, 11). Indeed, Mt-Lig progressively digested the 3′ but not the 5′ ss tails of partial duplexes (18) until reaching the double-strand (ds) region (Fig. 1B). Thus, Mt-Lig possesses 3′ to 5′ ssDNA exonuclease activity. Using DNA substrates that generate a 3′-flap adjacent to a nick, Mt-Lig removed the flap by exonucleolytic digestion, generating a base-paired linear duplex (Fig. 1C). At higher concentrations, the nuclease progressed through the microhomology region and into the duplex (Fig. 1C and fig. S4). Nuclease activity required the presence of magnesium or manganese. Mutation of a conserved histidine residue abolished this exonuclease activity, confirming that it is also an intrinsic property of Mt-Lig (fig. S5).

We thus asked whether this single polypeptide could repair a DSB that requires 3′ resection, gap filling, and ligation. In the presence of NTPs, Mt-Lig joined aligned DNA duplexes possessing a 1-nt 3′ flap adjacent to a 3-nt gap (Fig. 2A and fig. S6). A similar, albeit less efficient, reaction was observed in the presence of dNTPs (fig. S6). Neither the nuclease nor polymerase mutants were able to repair this junction, confirming that both activities were required to process the DSB (fig. S7). Sequencing of ligated junctions in equivalent assays with substrates with a 3-nt flap adjacent to a 5-nt gap revealed that the microhomology sequence was retained and that the mismatched flap was replaced by nucleotides complementary to the template strand (18).

Fig. 2.

Joining of DNA molecules with incompatible ends by Mt NHEJ. (A) Mt-Lig (1 μM) and the indicated DNA duplex were incubated with MgCl2, NTPs, and ATP for increasing times (minutes). S, DNA substrate alone. The positions of the 49-nt oligomer (substrate), 48-nt oligomer (removal of flap), 51-nt oligomer (gap fill-in product), and 92-nt oligomer (ligated product) are indicated. Asterisks denote labeled strands. (B) Effect of Mt-Ku on the nuclease activity of Mt-Lig. DNA duplexes were incubated with 1 μM Mt-Lig and increasing amounts of Mt-Ku (0.8, 1.6, 3.2, and 6.4 μM) without ATP. (C) Plasmid DNA cut with two restriction enzymes was incubated with Mt-Lig (4 pmol) and increasing amounts of Mt-Ku (0.05, 0.1, 0.5, and 1 pmol), with either dNTPs or NTPs, followed by PCR to monitor the rate of repair (bottom). Slower migrating products correspond to uncut plasmid, and the fastest migrating products (white arrows) correspond to successfully repaired junctions that have lost ∼500 base pairs between the restriction sites. pUC18, plasmid DNA. (D) Plasmids from experiments similar to (C) were transformed into bacteria and sequenced. The starting ends, final products, and inferred alignment intermediates are shown.

Mt-Ku stimulates joining of fully complementary ends by Mt-Lig (3). At incompatible ends, Mt-Ku did not have a significant effect on the removal of mismatched flaps, but it did inhibit further digestion into the microhomology region (Fig. 2B), suggesting that Mt-Ku remains physically associated with this region during repair. Using an in vitro polymerase chain reaction (PCR)–based plasmid repair assay (19), we observed that Mt-Ku markedly stimulated the joining of long linear DNA molecules with different incompatible ends (Fig. 2C). This occurred in the presence of either dNTPs or NTPs (Fig. 2C). In contrast, we observed no rejoining by T4 ligase in the presence or absence of Mt-Ku (Fig. 2C). Joining of partially complementary 5′ (Hind III–Nhe I) and 3′ (Pst I–Kpn I) overhangs appeared to require microhomology-mediated alignments that need gap filling and, in some instances, 3′ flap removal on one strand (Fig. 2D). Joining of a blunt end–3′ ss overhang (Sma I–Aat II) appeared to require the addition of one nucleotide by the terminal transferase activity, followed by microhomology pairing with the 3′ overhang, flap resection, gap filling, and ligation (Fig. 2D). In all cases, gap filling accurately copied the template strand (18).

To examine the rejoining of chromosome breaks in vivo, we exploited a variant of the yeast-based “suicide deletion” assay (20, 21), which allows the simultaneous determination of NHEJ and recombination frequencies (fig. S8A). Consistent with prior observations (20, 21), ∼75% of wild-type yeast cells repaired an I-SceI endonuclease-induced DSB by recombination and ∼2% by NHEJ, with the remainder dying (Fig. 3A). NHEJ occurred predominantly by simple religation [resulting in Ade+ colonies (fig. S8)] and was decreased 99% by yku70 (Ku) deletion. Introducing plasmids that express Mt-Ku and Mt-Lig restored NHEJ to about half its level in wild-type yeast (Fig. 3A and fig. S9A). Combinations of Mt-Ku, Mt-Lig, and yku70 and dnl4 (yeast ligase IV) mutations demonstrated that Mt NHEJ was truly reconstituted by a concerted species-specific Ku-ligase interaction (Fig. 3B).

Fig. 3.

Reconstitution of NHEJ in yeast by Mt proteins. (A) Frequencies of gene conversion and simple religation NHEJ in wild-type and yku70 mutant yeast demonstrate reconstitution of NHEJ by combined expression of Mt-Ku and Mt-Lig. wt, wild-type. (B) All combinations of yeast and bacterial Ku and ligase genes were tested for NHEJ function. Labels indicate those proteins that were present in the cell. (C) NHEJ catalyzed by Mt proteins is only partially dependent on an intact MRX complex. No Ade+ colonies were recovered from dnl4 rad50 yeast with vectors only.

In Saccharomyces cerevisiae, NHEJisalso dependent on the MRX complex (21, 22). MRX may act as an end-bridging factor and/or functionally interact with yeast Ku and Dnl4 (6, 23). Expression of the Mt NHEJ proteins in yeast rad50 mutants substantially recovered NHEJ (Fig. 3C), although to a lesser extent than seen with yku70 or dnl4 mutants. Thus, Mt NHEJ can occur in the absence of both MRX and its bacterial ortholog SbcCD (23).

As with NHEJ mediated by yeast proteins (20, 21, 24), Mt NHEJ reconstituted in yeast occasionally resulted in imperfect repair, evident as Ade colonies in the absence of the gene conversion donor. Sequencing of these colonies revealed a variety of junctions that occurred predominantly through mispairing of the I-SceI 3′ overhangs (fig. S9A). To create a suicide deletion system that selected specifically for such NHEJ events, we substituted HO (endonuclease) for I-SceI so that joints with a relative reading frame of +2 nucleotides yielded Ade+ colonies (fig. S8). Again consistent with previous results (16, 25), ∼0.75% of all NHEJ events in wild-type yeast were Ade+ (Fig. 4A), and >50% of these were HO(+2) joints (fig. S9). With Mt NHEJ reconstituted, the overall frequency of NHEJ remained high (18), but the percentage of Ade+ events was substantially decreased (Fig. 4A). Although some HO(+2) processed joints were formed, the HO(–1) joint now predominated (Fig. 4B), providing a signature for Mt NHEJ (Fig. 4C and fig. S9). Mt NHEJ proteins shifted the NHEJ profile even in wild-type yeast (Fig. 4, A and C, and fig. S9). Mt-Ku and Mt-Lig proteins can therefore catalyze processed NHEJ in chromosomes, but despite this ability, repair is highly accurate at compatible DSB ends.

Fig. 4.

Mt NHEJ exhibits a different pattern of imprecise NHEJ than yeast NHEJ. (A) The extent of +2 frame-shifted NHEJ was determined as a fraction of all NHEJ events. Mt NHEJ showed a markedly lower +2 frequency than yeast NHEJ. No Ade+ colonies recovered from yku70 yeast with vectors only. (B) Inferred NHEJ intermediates for the HO(+2) and HO(–1) events that give a +2 reading frame. (C) Genomic DNA was prepared from bulk cultures of the indicated yeast bearing the HO suicide deletion system after outgrowth in adenineselective medium. Flanking PCR was performed, and products were electrophoresed alongside standards derived from colonies of known joint types.

With the exception of 5′ trimming, Mt-Ku and Mt-Lig thus encode a self-sufficient NHEJ repair machine. The accuracy of this machine suggests that its different activities must be highly regulated to correctly handle different end configurations. Eukaryotic NHEJ involves many more proteins (1, 2), but we suggest that the mechanisms of end-processing will be fundamentally similar, although coordinated by protein-protein interactions rather than linkage in a single polypeptide. Indeed, the variety of nucleotidyl transferase activities in Mt-Lig suggests a reduced dependence on template similar to that of mammalian Pol μ and Pol λ (2628), which likely accommodates the limited base pairing inherent to NHEJ. As with Pol μ, Mt-Lig also appears most likely to incorporate NTPs in vivo, which has been suggested to be beneficial given the relative concentrations of NTPs and dNTPs outside of S phase (17). Incorporated ribonucleotides could be replaced by described mechanisms (29). Finally, Mt proteins catalyze NHEJ in yeast even though prokaryotes lack histones. This might suggest that a specific histone interaction is not required during NHEJ.

Supporting Online Material

www.sciencemag.org/cgi/content/full/306/5696/683/DC1

Materials and Methods

Figs. S1 to S9

References and Notes

References and Notes

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