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Mycobacteriophage Exploit NHEJ to Facilitate Genome Circularization
Molecular Cell 23, 743–748, September 1, 2006 ª2006 Elsevier Inc.
DOI 10.1016/j.molcel.2006.07.009
Mycobacteriophage Exploit NHEJ to Facilitate Genome Circularization Robert S. Pitcher,1 Louise M. Tonkin,1,2 James M. Daley,3 Phillip L. Palmbos,3 Andrew J. Green,1 Tricia L. Velting,3 Anna Brzostek,4 Malgorzata Korycka-Machala,4 Steve Cresawn,5 Jaroslaw Dziadek,4 Graham F. Hatfull,5 Thomas E. Wilson,3 and Aidan J. Doherty1,* 1 Genome Damage and Stability Centre University of Sussex Falmer, Brighton BN1 9RQ 2 Cambridge Institute for Medical Research Department of Haematology University of Cambridge Hills Road Cambridge CB2 2XY United Kingdom 3 Department of Pathology University of Michigan Medical School Ann Arbor, Michigan 48109 4 Centre of Medical Biology Polish Academy of Sciences 93-232 Lodz Lodowa 106 Poland 5 Pittsburgh Bacteriophage Institute Department of Biological Sciences University of Pittsburgh Pittsburgh, Pennsylvania 15260
Short Article
heterodimer, comprising two subunits (Ku70 and Ku80), is a vital component of the eukaryotic NHEJ repair complex involved in break recognition, end alignment, and recruitment of other repair factors, including DNA ligase IV, which ultimately seals the breaks (Doherty and Jackson, 2002; Walker et al., 2001). More recently, it has been reported that a related NHEJ pathway is also utilized by many species of bacteria, which depend on a prokaryotic Ku homolog and a distinct ATP-dependent DNA ligase (Bowater and Doherty, 2006; Della et al., 2004; Gong et al., 2004, 2005; Pitcher et al., 2005a, 2005b; Weller et al., 2002). Viruses and bacteriophage are obligate parasites that exploit host cell functions, including DNA replication and repair, for propagation. It is thus intriguing that NHEJ proteins, including Ku, have also been reported to play a role in the integration of retroviral DNA in eukaryotes (Daniel et al., 1999, 2004; Jeanson et al., 2002; Kilzer et al., 2003; Li et al., 2001; Skalka and Katz, 2005), whereas retrotransposition of the retroviral-like yeast transposon Ty is reliant on Ku (Downs and Jackson, 1999). The precise role of Ku and NHEJ in these processes remains to be established (Skalka and Katz, 2005). Here, we provide unification of these ideas by showing that NHEJ-dependent circularization of selected mycobacteriophage genomes is an essential part of their life cycle, establishing a new paradigm of host cell DNA repair function in viral infection. Results and Discussion
Summary Ku-dependent nonhomologous end joining (NHEJ) is a double-strand break repair process conserved in all branches of cellular life but has not previously been implicated in the DNA metabolic processes of viruses. We identified Ku homologs in Corndog and Omega, two related mycobacteriophages of Mycobacterium smegmatis. These proteins formed homodimers and bound DNA ends in a manner identical to other Ku’s and stimulated joining of ends by the host NHEJ DNA ligase (LigD). Omega and Corndog are unusual in having short 4 base cos ends that would not be expected to self-anneal and would therefore require NHEJ during phage genome circularization. Consistently, M. smegmatis LigD null strains are entirely and selectively unable to support infection by Corndog or Omega, with concomitant failure of genome circularization. These results establish a new paradigm for sequestration of the host cell NHEJ process by bacteriophage and provide a framework for understanding similar transactions in eukaryotic viral infections. Introduction The process of NHEJ is the major repair pathway responsible for the resolution of DNA double-strand breaks (DSBs) in higher eukaryotes (Krejci et al., 2003). The Ku *Correspondence: [email protected]
Mycobacteriophage Omega and Corndog Contain Functional Ku Homologs Pedulla et al. reported that the mycobacteriophage Omega and Corndog contain open reading frames (ORFs) with homology to the Rv0937c protein of Mycobacterium tuberculosis (Mt) (Pedulla et al., 2003). Rv0937c (Mt-Ku) is the M. tuberculosis Ku homolog (Weller et al., 2002). It binds directly to DSB ends, where it recruits the Mt NHEJ ligase (Mt-LigD) (Weller et al., 2002), which subsequently catalyses resection and ligation of DSBs (Bowater and Doherty, 2006; Della et al., 2004; Pitcher et al., 2005a, 2005b; Weller et al., 2002). To validate the presence of bacterial Ku-like proteins in mycobacteriophage, we performed iterative PHIBLAST database searches with the sequences of the phage ORFs Omega gp206 and Corndog gp87. The viral proteins were highly homologous with other members of the bacterial Ku family (Figure S1 in the Supplemental Data available with this article online). For example, BLAST searches with gp206 and gp87 detect Mt-Ku with Expect (E) values of E = 10270 and E = 10236, respectively. In line with these observations, the sequence-threading algorithm PHYRE (Kelley et al., 2000) predicted that both gp206 and gp87 (E = 10219 and 100% confidence) adopt secondary structures similar to the core DNA binding region of human Ku70 (Walker et al., 2001; Figure S2). Based on this and data presented below, we have renamed these proteins Omega Ku (U-Ku; gp206) and Corndog Ku (Cd-Ku; gp87). The
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close sequence homology between the mycobacteriophage and mycobacterial Ku’s (Figure S1), in conjunction with the absence of phage LigD homologs (see below), suggests that the phage Ku’s were probably acquired from the host by a lateral gene transfer event. Although other phage, including bacteriophage Mu, contain Ku-like proteins called Gam (d’Adda di Fagagna et al., 2003), these proteins form a distinct family of Ku proteins only distantly related to the eukaryotic, bacterial, and mycobacteriophage Ku’s. The mycobacteriophage U- and Cd-Ku genes were cloned, and recombinant histidine-tagged versions of the proteins were overexpressed in Escherichia coli and purified by nickel-Sepharose affinity and ion-exchange chromatography (Figure S3). Threading studies predicted that the mycobacteriophage Ku’s are homodimeric complexes, forming ring-like structures through which the DNA end is bound. Indeed, gel-filtration analysis of the purified phage Ku’s indicated that these proteins, in common with the bacterial Ku’s (Weller et al., 2002), exist as stable homodimers in solution (Figure S4). Again in common with the bacterial and eukaryotic Ku’s (d’Adda di Fagagna et al., 2003; Weller et al., 2002), U- and Cd-Ku bound to double-stranded (ds) DNA ends with high affinity (Figure 1), irrespective of overhang type, as determined by electrophoretic mobility shift assays (EMSAs), and protected DNA ends from exonuclease digestion (Figure S5). Furthermore, multiple mycobacteriophage Ku proteins can load onto a single DNA molecule in a ‘‘beads on a string’’ manner, suggesting that these proteins bind to DNA in a similar way to other Ku’s (Bliss and Lane, 1997; Weller et al., 2002). Mycobacteriophage Ku’s Stimulate End Joining by the Host NHEJ DNA Ligase Having verified that mycobacteriophage Ku’s share many of the biochemical properties of other Ku family members, we set out to establish the role, if any, played by these proteins in the phage life cycle. Corndog and Omega are each dsDNA viruses of the lambda phage family (Pedulla et al., 2003). The spectrum of bacteria they can infect is not known, but they were identified from environmental samples by using Mycobacterium smegmatis (Ms) as the obligate host (Pedulla et al., 2003), a bacterium known to possess Ku (Ms-Ku), LigD (Ms-LigD), and a functional NHEJ pathway (Gong et al., 2005; Korycka-Machala et al., 2006). This, and the striking sequence similarity between the mycobacteriophage and mycobacterial Ku proteins, thus led us to investigate if they showed a functional overlap in NHEJ. To identify if mycobacteriophage contained a Ku-associated ligase, we searched the Omega and Corndog genomes but, surprisingly, DNA ligase genes are absent. This is unusual, as most phage encode a gene for this class of enzyme. There are many examples where viruses compensate for lack of particular genes by usurping host proteins. To address this possibility, we tested if the phage Ku could recruit the host LigD and stimulate end joining. Using in vitro PCR-based plasmid repair assays (Della et al., 2004), we observed that both U- and Cd-Ku significantly stimulated the end-joining activity of Ms-LigD (Figure 2A). In all cases, the ability of U- or Cd-Ku to stimulate Ms-LigD in vitro was quantitatively
Figure 1. U-Ku and Cd-Ku Bind dsDNA Electrophoretic-mobility shift assays were employed to characterize the ability of (A) U-Ku and (B) Cd-Ku to bind DNA. Reaction mixtures contained 10 nM labeled DNA duplex (with blunt-ended or with 30 or 50 overhangs as illustrated). Lane 1, labeled DNA only; and lanes 2–6, increasing amounts of protein added to DNA (0.05, 0.125, 0.25, 0.5, and 0.75 mM). (C) U- and Cd-Ku also super shifted linearized pUC18 plasmid DNA separated on agarose gels. Reaction mixtures contained dsDNA (digested with EcoRI) and (0–15 mM) protein. Lane 1, DNA alone; and lanes 2–6, increasing amounts of protein added to DNA (1, 2, 5, 10, and 15 mM).
similar to Ms-Ku, suggesting this as a bona fide action of these proteins. We next looked for potential interactions between MsLigD and the phage Ku’s by EMSAs with a radio-labeled dsDNA probe (33 bp). Ms-LigD alone did not produce a significant band-shift complex, suggesting that it has weak affinity for DNA ends (Figure 2B). In contrast, the inclusion of either U- or Cd-Ku to EMSAs containing Ms-LigD resulted in the generation of super-shifted complexes with higher mobility, distinct from complexes formed by either Ku protein alone (Figure 2B). The formation of the super-shifted species did not occur if Ms-LigD was denatured, indicating that it reflects the binding of Ms-LigD. Previously, we reported that the interaction between Mt-Ku and LigD is mediated through the polymerase domain (PolDom) of Mt-LigD (Pitcher et al., 2005a), and EMSAs showed that U-Ku also interacts with the equivalent PolDom of Ms-LigD (Figure 2B). Mycobacteriophage Ku’s and Bacterial Ligase D Together Function as an NHEJ Repair Complex In Vivo The above results strongly suggest that the phage Ku’s are involved in NHEJ that utilizes the host cell NHEJ ligase. To demonstrate this ability in living cells, we
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Figure 3. U-Ku Cooperates with Mycobacterial Ligase D to Reconstitute NHEJ in Yeast
Figure 2. U-Ku and Cd-Ku Recruit and Stimulate Ms-LigD (A) Complementary end joining by Ms-LigD in the presence of U- and Cd-Ku. Lane 1, 60 nM U- or Cd-Ku; and lanes 2–7, 20 nM Ms-LigD with 0, 10, 20, 40, 80, and 125 nM U- or Cd-Ku; and lane 8, 20 nM Ms-LigD and 20 nM Ms-Ku. (B) Ms-LigD and Ms-PolDom super shifted both the U- and Cd-Ku’s and the U-Ku, respectively. Reaction mixtures contained 10 nM labeled dsDNA 33 bp duplex. Lane 1, labeled probe only; lane 2, 0.8 mM U-Ku; lane 3, 0.8 mM Cd-Ku; lane 4, 0.5 mM Ms-PolDom; lanes 5–7, 0.2, 0.4, and 0.8 mM U-Ku in addition to Ms-PolDom (0.5 mM); lane 8, 0.5 mM Ms-LigD; lanes 9–11, 0.2, 0.4, and 0.8 mM U-Ku in addition to Ms-LigD (0.5 mM); and lanes 12–14, 0.2, 0.4, and 0.8 mM Cd-Ku in addition to Ms-LigD (0.5 mM).
exploited yeast-based assays previously used to document the functional link between Mt-Ku and Mt-LigD (Della et al., 2004). Various combinations of phage and bacterial Ku and LigD were expressed in Saccharomyces cerevisiae made NHEJ deficient by host yku70 (Ku) and dnl4 (ligase IV) mutations. We first transformed these yeasts with plasmids linearized in the MET15 marker gene by restriction enzymes that generated fully compatible 4 base overhangs with 50 or 30 polarity. Transformation to Met+ by these plasmids, which requires repair of the DSB by NHEJ, was observed above background levels only when both ectopic Ku and ligase
Bacterial and phage Ku and LigD proteins were expressed in dnl4 yku70 yeast strains lacking endogenous NHEJ in the indicated combinations, and NHEJ efficiency was examined by plasmid recircularization and chromosomal suicide deletion assays as described in the Experimental Procedures. (A) Plasmid DSBs were created with either NsiI (4 base 30 overhangs, top) or NcoI (4 base 50 overhangs, bottom). NHEJ capacity is expressed as the Met+ transformation efficiency by these plasmids normalized to the His+ transformation efficiency by a cotransformed circular HIS3-marked plasmid. (B) Chromosomal DSBs in ADE2 were created by expressing the mega-endonuclease I-SceI (4 base 30 overhangs). NHEJ capacity is expressed as the fraction of cells that survived I-SceI induction as Ade+ (w1% for wild-type yeast). Results are plotted on a log scale. Each bar represents the mean 6 standard deviation (error bar) from three independent experiments. For each assay, U-Ku stimulated LigD-dependent NHEJ, although to different extents in plasmid and chromosomal assays as compared to Mt-Ku and Ms-Ku.
were expressed (Figure 3A), demonstrating that the proteins must act together. U-Ku and the bacterial Ku’s could each stimulate joining by both Mt- and Ms-LigD (Figure 3A), demonstrating that the phage and bacterial Ku’s were redundant and promiscuous in their ability to recruit LigD across bacterial species boundaries. We also employed an assay in which a chromosomal DSB is made in vivo in the ADE2 marker by the endonuclease I-SceI (Della et al., 2004; Karathanasis and Wilson, 2002), where only NHEJ can accurately repair the DSB to Ade+. Repair by LigD was again facilitated by U-Ku, although here the effect was not as pronounced as with Ms-Ku and Mt-Ku (Figure 3B). These results establish that, like the bacterial Ku proteins, U-Ku can function with LigD in NHEJ of single DSBs in the absence of other phage or bacterial proteins.
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Mycobacteriophage Omega and Corndog Utilize NHEJ to Facilitate Genome Circularization The ability of U- and Cd-Ku to facilitate LigD-dependent NHEJ predicted a role for NHEJ in the bacteriophage life cycle that, given that such a role was previously undocumented, in turn predicted that Omega and Corndog must have atypical features. Lambda-like dsDNA bacteriophage typically enter the cell as linear genomes, which are promptly circularized by self-association of long (R9 bases) complementary overhangs called cohesive (cos) ends (Ellis and Dean, 1985). We recently described the profile of rejoining of overhangs of varying length in yeast and showed that this length is a critical parameter in determining the repair mechanism, with repair becoming strictly Ku and NHEJ dependent when overhangs became shorter than six bases (Daley and Wilson, 2005). As genome circularization was a strong candidate for a phage Ku function, we hypothesized that Omega and Corndog would have short cos overhangs. Indeed, comparison of sequences obtained directly from the linear genome with sequences obtained from the genome circularized by ligation confirmed that both Omega and Corndog have 4 base 30 complementary overhanging ends (Figure S6). In contrast, other lambda-like mycobacteriophage described in the same studies as Corndog and Omega (Pedulla et al., 2003), but lacking a phage Ku homolog, showed more typical nine or ten base overhangs expected to show a favorable association equilibrium (Ellis and Dean, 1985). Only circular genomes can support rolling circle replication used to make progeny phage and maintain infectivity. Abolition of NHEJ should therefore lead to lost infectivity by Corndog and Omega, if the circularization hypothesis is correct. To test this, we utilized M. smegmatis strains deficient in Ku (DKu), LigD (DLigD), and a double-knockout strain (DKu, DLigD) (Korycka-Machala et al., 2006). Infection of wild-type M. smegmatis with either Omega or Corndog phage resulted in significant plaque formation on plates (Figure 4A). A similar level of infection was also observed in the DKu strain, consistent with the interpretation that the phage Ku is able to functionally replace the host Ku in phage genome NHEJ, as was observed in the yeast plasmid experiments. However, in a DLigD strain, as well as in a DKu, DLigD strain, absolutely no plaques were formed (Figure 4A), demonstrating that host LigD is essential for phage propagation. To confirm that this phenotype was due to LigD itself, we reintroduced Ms-LigD on an acetamide-inducible plasmid. This genetic complementation resulted in a complete restoration of plaque formation by both Omega and Corndog in the DLigD strain (Figure 4B). Importantly, LigD is a multifunctional protein, but our model predicts that the ligation activity of LigD will be specifically essential for plaque production. To test this, we mutated Ms-LigD gene at two regions (Figure S7), replacing the essential ligase active site lysine with alanine (K484A; ligase domain) and substituting conserved polymerase aspartate residues with alanine (D136A, D138A; PolDom). Although M. smegmatis DLigD strains, transformed with plasmids carrying either the wild-type Ms-LigD or Ms-LigD (D136A, D138), supported infection and plaque formation by Omega and Corndog, there was a clear absence of plaques in strains
Figure 4. Ms-LigD, but Not Ms-Ku, Is Required for Omega and Corndog Plaque Formation in M. smegmatis (A) Plaques formed when either wild-type or DKu M. smegmatis strains were infected with Omega or Corndog phage lysate. However, no plaques formed when either DLigD or DLigD, DKu strains were infected with Omega or Corndog phage lysate. (B) The DNA ligase, but not the polymerase, activity of Ms-LigD was required for plaque formation. Plaques appeared when the LigD deletion strain is complemented with Ms-LigD expressed from an acetamide-inducible plasmid. Plaques also formed when the LigD deletion strain was complemented with Ms-LigD (containing catalytic mutants that prevent polymerase activity) were infected with either phage lysate. However, plaques did not appear when a LigD deletion strain complemented with Ms-LigD (containing a catalytic mutation [K484A] that prevented ligase activity) was infected with either phage lysate.
carrying plasmid pLigD (K484A) (Figure 4B). Thus, the ligation activity of the host NHEJ ligase is indeed essential for the life cycle of these mycobacteriophage. In contrast, the polymerase activity of Ms-LigD is dispensable (Figure 4B), presumably because the genome ends are generated by the observed Corndog and Omega terminase (Pedulla et al., 2003). Terminase-generated ends are expected to be uniformly complementary and require no processing prior to ligation, as typical for all bacteriophage. A corollary is that the NHEJ process that circularizes Corndog and Omega must be predominantly accurate, without nucleotide additions and deletions, so that subsequent cleavage of the cos region by terminase during rolling circle replication can proceed and create the uniform ends observed in the genome (Figure S6). This is seemingly distinct from plasmid transformation studies of M. smegmatis, which have suggested that its NHEJ is highly imprecise (Gong et al., 2005). PCR analysis of phage genomic DNA extracted from bacteria infected with either Omega or
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Corndog phage confirmed the presence of closed-circular viral genomic DNA species in wild-type M. smegmatis. (Figure S8). Finally, our model predicts that infection by phage with longer overhangs should be NHEJ, and therefore LigD, independent. Indeed, mycobacteriophage D29 (Pedulla et al., 2003), which contains a nine base overhanginfected M. smegmatis (DLigD) as readily as the wildtype strain (Figure S9), additionally demonstrating that this strain is not deficient in supporting phage replication when NHEJ-dependent circularization is not required. Conclusions We have presented data demonstrating that selected mycobacteriophage of the lambda family depend on the host-encoded NHEJ ligase for circularization of their dsDNA genomes, in a manner that correlates precisely with the shortness of their cos overhangs. LigD’s essential function is ligation, as evidenced by selective point mutants. The host Ku is not required apparently because these bacteriophage, Corndog and Omega, encode their own Ku homolog, which interacts functionally with the host LigD in vitro and can activate it for NHEJ in ectopic NHEJ reconstitution in vivo. In addition to facilitating ligation, bacteriophage Ku may also serve to specifically protect the phage genomic ends from degradation upon cellular entry (Figure S5), providing an explanation for the existence of a phage-encoded Ku’s as opposed to sequestration of the entire cellular NHEJ machinery (Bowater and Doherty, 2006; d’Adda di Fagagna et al., 2003; Pitcher et al., 2005b). Taken together, our results emphasize that circularization of dsDNA genomes of viruses, and indeed all ‘‘repair’’ steps of viral life cycles, are subject to the same rules and conditions as chromosomal DSBs, and provide a framework for understanding the importance of DSB repair processes in viral infection. For example, the dsDNA genome of herpes simplex virus type 1 (HSV-1) can be circularized (Mocarski and Roizman, 1982), although it is uncertain whether this is a required step in lytic infection (Strang and Stow, 2005). Our results point to the possibility that such circularization events may indeed be a normal part of the viral life cycle and that NHEJ might be expected to play an important role in this process given its predominance in eukaryotic cells. Interestingly, the HSV protein vmw110 induces the degradation of the catalytic subunit of DNA-dependent protein kinase (Parkinson et al., 1999), but the effect of this degradation on the balance of apoptotic signaling and repair of viral DSB ends is not established. In a different viral type, HIV genomes can be circularized after reverse transcription into dsDNA by at least two mechanisms that preserve one or both of the long terminal repeats (LTRs) (Jeanson et al., 2002; Kilzer et al., 2003; Li et al., 2001; Skalka and Katz, 2005). The fact that Ku deficiency impairs only circularization that preserves both LTRs suggests that this represents NHEJ, whereas one-LTR circularization may perhaps be analogous to single-strand annealing (Ivanov et al., 1996). Such processes may have substantial indirect effects on the ability of a cell to support infection, for example by leading to persistence of DSB ends or altered damage signaling, in contrast to the suggested direct role of NHEJ in retroviral integration (Skalka and Katz, 2005).
Experimental Procedures Expression Constructs Full-length sequences for Ms-LigD and Ms-Ku were amplified by PCR from M. smegmatis mc2155 genomic DNA. Omega-Ku (gp206) and Corndog-Ku (gp87) were amplified from phage genomic DNA. All PCR products were cloned into pET28a (Novagen). Ms-PolDom was constructed by replacing R300 (Ms-LigD numbering) with a stop codon using pMs-LigD as a template. Overexpression and Purification of Constructs Ms-PolDom and Ms-LigD were overexpressed and purified as described previously (Gong et al., 2005; Pitcher et al., 2005a). Recombinant Ku proteins (Ms-Ku, Omega-Ku, and Corndog-Ku) were purified as described previously (Weller et al., 2002). EMSAs EMSAs were conducted by using linear DNA duplexes as described previously (Weller et al., 2002). Plasmid Repair Assay Plasmid repair assays were performed as previously described (Della et al., 2004). One-hundred nanograms of PvuI-cut pUC18 and 0–125 nM U- or Cd-Ku, and when present Ms-Ku (20 nM), were incubated for 15 min. Ms-LigD (20 nM) was then added, and the reactions were incubated for 1 hr at 37 C. The reactions were examined by PCR, using forward and reverse primers upstream and downstream respectively of the restriction site. PCR products were separated on a 1% (w/v) agarose gel stained with ethidium bromide. Yeast NHEJ Assays Plasmid-based expression of Mt-Ku and Mt-LigD in yeast from the ADH1 promoter as Myc-NLS fusion proteins was as previously described (Della et al., 2004). Ms-Ku, Ms-LigD, and U-Ku were expressed similarly in various combinations. The yeast strain was YW1231 (Della et al., 2004), which was dnl4 yku70 mutant to make it NHEJ deficient, and bore the I-SceI suicide deletion allele for monitoring chromosomal repair (Karathanasis and Wilson, 2002). For plasmid assays, 100 ng of pRS411 cut with either NcoI or NsiI within its MET15 marker was cotransformed with 20 ng HIS3-marked pRS413 (Brachmann et al., 1998) as previously described (Daley and Wilson, 2005). Repair efficiency is expressed as the ratio of Met+ to His+ colonies. For chromosomal assays, yeast were plated to galactose medium to induce I-SceI expression. Repair efficiency is expressed as the ratio of Ade+ colonies on galactose, indicating repair by NHEJ, to total viable colonies on parallel glucose plates. Growth and Infection of Mutant M. smegmatis Strains Wild-type M. smegmatis and mutant strains were grown from single colonies at 37 C with shaking in Middlebrook 7H9 broth (Difco) supplemented with 7H9 ADC enrichment media (Difco) to an OD600 of w1. Complementation of DLigD strains required 0.2% acetamide in culture media to stimulate transcription of the LigD gene on the plasmid. If necessary, the ODs were adjusted, before 0.1 ml of each culture was mixed with 0.1 ml of tenfold serial dilutions (in SM buffer) of phage lysate and incubated at 37 C for 20 min. Then, 150 ml of this mixture was added to 7 ml of top agar (diluted 7H10 Middlebrook agar [Difco] supplemented with OADC enrichment media [Difco]) quickly mixed and plated out onto 7H10 Middlebrook agar supplemented with OADC and incubated at 37 C for 24–48 hr. To confirm that circularization was part of the phage life cycle, primer pairs were designed for the 50 and 30 termini of either the Omega or Corndog genome that would produce a 500 bp fragment by PCR if the genomes were indeed circularized during plaque formation. Primers that would produce a 1 Kb fragment whether the genomes were linear or circular were also used as controls. Directed Mutagenesis of Ms-LigD Gene The key amino acids of the polymerase (136D, 138D) and ligase (484K) active sites of LigD were replaced with alanine (A) by using PCR-directed mutagenesis. The resultant PCR products were cloned into pGemT-easy vector and sequenced to confirm the introduced mutation (Figure S7). Finally, the mutated ligD genes were
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introduced into the pJam2 shuttle vector under the control of an acetamide promoter (Triccas et al., 1998). The resultant vectors pJam2-ligDmutL (ligase mutant) and pJam2-ligDmutP (polymerase mutant) were introduced into an M. smegmatis strain carrying an unmarked deletion in the wild-type ligD gene (M. smegmatis DligD) (Korycka-Machala et al., 2006). Supplemental Data Supplemental Data include nine figures and can be found with this article online at http://www.molecule.org/cgi/content/full/23/5/ 743/DC1/. Acknowledgments We thank N. Brissett, R. Curtis, E. Davis, M. Pedulla, C. Peebles, and J. van Kessel for their valuable assistance and comments. A.J.D. is a Royal Society University Research Fellow and A.J.D.’s laboratory is supported by grants from the Biotechnology and Biological Sciences Research Council, American Institute for Cancer Research, and Cancer Research-UK. T.E.W. is supported by grants from United States Public Health Service and the Pew Charitable Trusts. Received: April 25, 2006 Revised: June 21, 2006 Accepted: July 10, 2006 Published: August 31, 2006 References Bowater, R., and Doherty, A.J. (2006). Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining. PLOS Genet. 2, 93–99. 10.1371/journal.pgen.0020008. Bliss, T.M., and Lane, D.P. (1997). Ku selectively transfers between DNA molecules with homologous ends. J. Biol. Chem. 272, 5765– 5773. Brachmann, C.B., Davies, A., Cost, G.J., Caputo, E., Li, J., Hieter, P., and Boeke, J.D. (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132. d’Adda di Fagagna, F., Weller, G.R., Doherty, A.J., and Jackson, S.P. (2003). The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku. EMBO Rep. 1, 47–53. Daley, J.M., and Wilson, T.E. (2005). Rejoining of DNA double-strand breaks as a function of overhang length. Mol. Cell. Biol. 25, 896–906. Daniel, R., Katz, R.A., and Skalka, A.M. (1999). A role for DNA-PK in retroviral DNA integration. Science 284, 644–647. Daniel, R., Greger, J.G., Katz, R.A., Taganov, K.D., Wu, X., Kappes, J.C., and Skalka, A.M. (2004). Evidence that stable retroviral transduction and cell survival following DNA integration depend on components of the nonhomologous end joining repair pathway. J. Virol. 78, 8573–8581. Della, M., Palmbos, P.L., Tseng, H.M., Tonkin, L.M., Daley, J.M., Topper, L.M., Pitcher, R.S., Tomkinson, A.E., Wilson, T.E., and Doherty, A.J. (2004). Mycobacterial Ku and ligase proteins constitute a two-component NHEJ repair machine. Science 306, 683–685. Doherty, A.J., and Jackson, S.P. (2002). How Ku makes ends meet. Curr. Biol. 11, R920–R924. Downs, J.A., and Jackson, S.P. (1999). Involvement of DNA endbinding protein Ku in Ty element retrotransposition. Mol. Cell. Biol. 19, 6260–6268. Ellis, D.M., and Dean, D.H. (1985). Nucleotide sequence of the cohesive single-stranded ends of Bacillus subtilis temperate bacteriophage phi 105. J. Virol. 55, 513–515. Gong, C., Martins, A., Bongiorno, P., Glickman, M., and Shuman, S. (2004). Biochemical and genetic analysis of the four DNA ligases of mycobacteria. J. Biol. Chem. 279, 20594–20606. Gong, C., Bongiorno, P., Martins, A., Stephanou, N.C., Zhu, H., Shuman, S., and Glickman, M.S. (2005). Mechanism of nonhomologous end-joining in mycobacteria: A low-fidelity repair system driven by Ku, ligase D and ligase C. Nat. Struct. Mol. Biol. 12, 304–312.
Ivanov, E.L., Sugawara, N., Fishman-Lobell, J., and Haber, J.E. (1996). Genetic requirements for the single-strand annealing pathway of double-strand break repair in Saccharomyces cerevisiae. Genetics 142, 693–704. Jeanson, L., Subra, F., Vaganay, S., Hervy, M., Marangoni, E., Bourhis, J., and Mouscadet, J.F. (2002). Effect of Ku80 depletion on the preintegrative steps of HIV-1 replication in human cells. Virology 300, 100–108. Karathanasis, E., and Wilson, T.E. (2002). Enhancement of Saccharomyces cerevisiae end-joining efficiency by cell growth stage but not by impairment of recombination. Genetics 161, 1015–1027. Kelley, L.A., MacCallum, R.M., and Sternberg, M.J. (2000). Enhanced genome annotation using structural profiles in the program 3DPSSM. J. Mol. Biol. 299, 499–520. Kilzer, J.M., Stracker, T., Beitzel, B., Meek, K., Weitzman, M., and Bushman, F.D. (2003). Roles of host cell factors in circularization of retroviral DNA. Virology 314, 460–467. Korycka-Machala, M., Brzostek, A., Rozalska, S., Rumijowska-Galewicz, A., Dziedzic, R., Bowater, R., and Dziadek, J. (2006). Distinct DNA repair pathways involving RecA and non-homologous endjoining in Mycobacterium smegmatis. FEMS Microbiol. Lett. 258, 83–91. Krejci, L., Chen, L., Van Komen, S., Sung, P., and Tomkinson, A.E. (2003). Mending the break: two repair machines in eukaryotes. Prog. Nucleic Acid Res. Mol. Biol. 74, 159–201. Li, L., Olvera, J.M., Yoder, K.E., Mitchell, R.S., Butler, S.L., Lieber, M., Martin, S.L., and Bushman, F.D. (2001). Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection. EMBO J. 20, 3272–3281. Mocarski, E.S., and Roizman, B. (1982). Structure and role of the herpes simplex virus DNA termini in inversion, circularization and generation of virion DNA. Cell 31, 89–97. Parkinson, J., Lees-Miller, S.P., and Everett, R.D. (1999). Herpes simplex virus type 1 immediate-early protein vmw110 induces the proteasome-dependent degradation of the catalytic subunit of DNA-dependent protein kinase. J. Virol. 73, 650–657. Pedulla, M.L., Ford, M.E., Houtz, J.M., Karthikeyan, T., Wadsworth, C., Lewis, J.A., Jacobs-Sera, D., Falbo, J., Gross, J., Pannunzio, N.R., et al. (2003). Origins of highly mosaic mycobacteriophage genomes. Cell 113, 171–182. Pitcher, R.S., Tonkin, L.M., Green, A.J., and Doherty, A.J. (2005a). Domain structure of a NHEJ DNA repair ligase from Mycobacterium tuberculosis. J. Mol. Biol. 351, 531–544. Pitcher, R.S., Wilson, T.E., and Doherty, A.J. (2005b). New insights into NHEJ repair processes in prokaryotes. Cell Cycle 4, 675–678. Skalka, A.M., and Katz, R.A. (2005). Retroviral DNA integration and the DNA damage response. Cell Death Differ. 12, 971–978. Strang, B.L., and Stow, N.D. (2005). Circularization of the herpes simplex virus type 1 genome upon lytic infection. J. Virol. 79, 12487–12494. Triccas, J.A., Parish, T., Britton, W.J., and Gicquel, B. (1998). An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis. FEMS Microbiol. Lett. 167, 151–156. Walker, J.R., Corpina, R.A., and Goldberg, J. (2001). Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 412, 607–614. Weller, G.R., Kysela, B., Roy, R., Tonkin, L.M., Scanlan, E., Della, M., Devine, S.K., Day, J.P., Wilkinson, A., d’Adda di Fagagna, F., et al. (2002). Identification of a DNA non-homologus end-joining complex in bacteria. Science 297, 1686–1689.
DOI 10.1016/j.molcel.2006.07.009
Mycobacteriophage Exploit NHEJ to Facilitate Genome Circularization Robert S. Pitcher,1 Louise M. Tonkin,1,2 James M. Daley,3 Phillip L. Palmbos,3 Andrew J. Green,1 Tricia L. Velting,3 Anna Brzostek,4 Malgorzata Korycka-Machala,4 Steve Cresawn,5 Jaroslaw Dziadek,4 Graham F. Hatfull,5 Thomas E. Wilson,3 and Aidan J. Doherty1,* 1 Genome Damage and Stability Centre University of Sussex Falmer, Brighton BN1 9RQ 2 Cambridge Institute for Medical Research Department of Haematology University of Cambridge Hills Road Cambridge CB2 2XY United Kingdom 3 Department of Pathology University of Michigan Medical School Ann Arbor, Michigan 48109 4 Centre of Medical Biology Polish Academy of Sciences 93-232 Lodz Lodowa 106 Poland 5 Pittsburgh Bacteriophage Institute Department of Biological Sciences University of Pittsburgh Pittsburgh, Pennsylvania 15260
Short Article
heterodimer, comprising two subunits (Ku70 and Ku80), is a vital component of the eukaryotic NHEJ repair complex involved in break recognition, end alignment, and recruitment of other repair factors, including DNA ligase IV, which ultimately seals the breaks (Doherty and Jackson, 2002; Walker et al., 2001). More recently, it has been reported that a related NHEJ pathway is also utilized by many species of bacteria, which depend on a prokaryotic Ku homolog and a distinct ATP-dependent DNA ligase (Bowater and Doherty, 2006; Della et al., 2004; Gong et al., 2004, 2005; Pitcher et al., 2005a, 2005b; Weller et al., 2002). Viruses and bacteriophage are obligate parasites that exploit host cell functions, including DNA replication and repair, for propagation. It is thus intriguing that NHEJ proteins, including Ku, have also been reported to play a role in the integration of retroviral DNA in eukaryotes (Daniel et al., 1999, 2004; Jeanson et al., 2002; Kilzer et al., 2003; Li et al., 2001; Skalka and Katz, 2005), whereas retrotransposition of the retroviral-like yeast transposon Ty is reliant on Ku (Downs and Jackson, 1999). The precise role of Ku and NHEJ in these processes remains to be established (Skalka and Katz, 2005). Here, we provide unification of these ideas by showing that NHEJ-dependent circularization of selected mycobacteriophage genomes is an essential part of their life cycle, establishing a new paradigm of host cell DNA repair function in viral infection. Results and Discussion
Summary Ku-dependent nonhomologous end joining (NHEJ) is a double-strand break repair process conserved in all branches of cellular life but has not previously been implicated in the DNA metabolic processes of viruses. We identified Ku homologs in Corndog and Omega, two related mycobacteriophages of Mycobacterium smegmatis. These proteins formed homodimers and bound DNA ends in a manner identical to other Ku’s and stimulated joining of ends by the host NHEJ DNA ligase (LigD). Omega and Corndog are unusual in having short 4 base cos ends that would not be expected to self-anneal and would therefore require NHEJ during phage genome circularization. Consistently, M. smegmatis LigD null strains are entirely and selectively unable to support infection by Corndog or Omega, with concomitant failure of genome circularization. These results establish a new paradigm for sequestration of the host cell NHEJ process by bacteriophage and provide a framework for understanding similar transactions in eukaryotic viral infections. Introduction The process of NHEJ is the major repair pathway responsible for the resolution of DNA double-strand breaks (DSBs) in higher eukaryotes (Krejci et al., 2003). The Ku *Correspondence: [email protected]
Mycobacteriophage Omega and Corndog Contain Functional Ku Homologs Pedulla et al. reported that the mycobacteriophage Omega and Corndog contain open reading frames (ORFs) with homology to the Rv0937c protein of Mycobacterium tuberculosis (Mt) (Pedulla et al., 2003). Rv0937c (Mt-Ku) is the M. tuberculosis Ku homolog (Weller et al., 2002). It binds directly to DSB ends, where it recruits the Mt NHEJ ligase (Mt-LigD) (Weller et al., 2002), which subsequently catalyses resection and ligation of DSBs (Bowater and Doherty, 2006; Della et al., 2004; Pitcher et al., 2005a, 2005b; Weller et al., 2002). To validate the presence of bacterial Ku-like proteins in mycobacteriophage, we performed iterative PHIBLAST database searches with the sequences of the phage ORFs Omega gp206 and Corndog gp87. The viral proteins were highly homologous with other members of the bacterial Ku family (Figure S1 in the Supplemental Data available with this article online). For example, BLAST searches with gp206 and gp87 detect Mt-Ku with Expect (E) values of E = 10270 and E = 10236, respectively. In line with these observations, the sequence-threading algorithm PHYRE (Kelley et al., 2000) predicted that both gp206 and gp87 (E = 10219 and 100% confidence) adopt secondary structures similar to the core DNA binding region of human Ku70 (Walker et al., 2001; Figure S2). Based on this and data presented below, we have renamed these proteins Omega Ku (U-Ku; gp206) and Corndog Ku (Cd-Ku; gp87). The
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close sequence homology between the mycobacteriophage and mycobacterial Ku’s (Figure S1), in conjunction with the absence of phage LigD homologs (see below), suggests that the phage Ku’s were probably acquired from the host by a lateral gene transfer event. Although other phage, including bacteriophage Mu, contain Ku-like proteins called Gam (d’Adda di Fagagna et al., 2003), these proteins form a distinct family of Ku proteins only distantly related to the eukaryotic, bacterial, and mycobacteriophage Ku’s. The mycobacteriophage U- and Cd-Ku genes were cloned, and recombinant histidine-tagged versions of the proteins were overexpressed in Escherichia coli and purified by nickel-Sepharose affinity and ion-exchange chromatography (Figure S3). Threading studies predicted that the mycobacteriophage Ku’s are homodimeric complexes, forming ring-like structures through which the DNA end is bound. Indeed, gel-filtration analysis of the purified phage Ku’s indicated that these proteins, in common with the bacterial Ku’s (Weller et al., 2002), exist as stable homodimers in solution (Figure S4). Again in common with the bacterial and eukaryotic Ku’s (d’Adda di Fagagna et al., 2003; Weller et al., 2002), U- and Cd-Ku bound to double-stranded (ds) DNA ends with high affinity (Figure 1), irrespective of overhang type, as determined by electrophoretic mobility shift assays (EMSAs), and protected DNA ends from exonuclease digestion (Figure S5). Furthermore, multiple mycobacteriophage Ku proteins can load onto a single DNA molecule in a ‘‘beads on a string’’ manner, suggesting that these proteins bind to DNA in a similar way to other Ku’s (Bliss and Lane, 1997; Weller et al., 2002). Mycobacteriophage Ku’s Stimulate End Joining by the Host NHEJ DNA Ligase Having verified that mycobacteriophage Ku’s share many of the biochemical properties of other Ku family members, we set out to establish the role, if any, played by these proteins in the phage life cycle. Corndog and Omega are each dsDNA viruses of the lambda phage family (Pedulla et al., 2003). The spectrum of bacteria they can infect is not known, but they were identified from environmental samples by using Mycobacterium smegmatis (Ms) as the obligate host (Pedulla et al., 2003), a bacterium known to possess Ku (Ms-Ku), LigD (Ms-LigD), and a functional NHEJ pathway (Gong et al., 2005; Korycka-Machala et al., 2006). This, and the striking sequence similarity between the mycobacteriophage and mycobacterial Ku proteins, thus led us to investigate if they showed a functional overlap in NHEJ. To identify if mycobacteriophage contained a Ku-associated ligase, we searched the Omega and Corndog genomes but, surprisingly, DNA ligase genes are absent. This is unusual, as most phage encode a gene for this class of enzyme. There are many examples where viruses compensate for lack of particular genes by usurping host proteins. To address this possibility, we tested if the phage Ku could recruit the host LigD and stimulate end joining. Using in vitro PCR-based plasmid repair assays (Della et al., 2004), we observed that both U- and Cd-Ku significantly stimulated the end-joining activity of Ms-LigD (Figure 2A). In all cases, the ability of U- or Cd-Ku to stimulate Ms-LigD in vitro was quantitatively
Figure 1. U-Ku and Cd-Ku Bind dsDNA Electrophoretic-mobility shift assays were employed to characterize the ability of (A) U-Ku and (B) Cd-Ku to bind DNA. Reaction mixtures contained 10 nM labeled DNA duplex (with blunt-ended or with 30 or 50 overhangs as illustrated). Lane 1, labeled DNA only; and lanes 2–6, increasing amounts of protein added to DNA (0.05, 0.125, 0.25, 0.5, and 0.75 mM). (C) U- and Cd-Ku also super shifted linearized pUC18 plasmid DNA separated on agarose gels. Reaction mixtures contained dsDNA (digested with EcoRI) and (0–15 mM) protein. Lane 1, DNA alone; and lanes 2–6, increasing amounts of protein added to DNA (1, 2, 5, 10, and 15 mM).
similar to Ms-Ku, suggesting this as a bona fide action of these proteins. We next looked for potential interactions between MsLigD and the phage Ku’s by EMSAs with a radio-labeled dsDNA probe (33 bp). Ms-LigD alone did not produce a significant band-shift complex, suggesting that it has weak affinity for DNA ends (Figure 2B). In contrast, the inclusion of either U- or Cd-Ku to EMSAs containing Ms-LigD resulted in the generation of super-shifted complexes with higher mobility, distinct from complexes formed by either Ku protein alone (Figure 2B). The formation of the super-shifted species did not occur if Ms-LigD was denatured, indicating that it reflects the binding of Ms-LigD. Previously, we reported that the interaction between Mt-Ku and LigD is mediated through the polymerase domain (PolDom) of Mt-LigD (Pitcher et al., 2005a), and EMSAs showed that U-Ku also interacts with the equivalent PolDom of Ms-LigD (Figure 2B). Mycobacteriophage Ku’s and Bacterial Ligase D Together Function as an NHEJ Repair Complex In Vivo The above results strongly suggest that the phage Ku’s are involved in NHEJ that utilizes the host cell NHEJ ligase. To demonstrate this ability in living cells, we
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Figure 3. U-Ku Cooperates with Mycobacterial Ligase D to Reconstitute NHEJ in Yeast
Figure 2. U-Ku and Cd-Ku Recruit and Stimulate Ms-LigD (A) Complementary end joining by Ms-LigD in the presence of U- and Cd-Ku. Lane 1, 60 nM U- or Cd-Ku; and lanes 2–7, 20 nM Ms-LigD with 0, 10, 20, 40, 80, and 125 nM U- or Cd-Ku; and lane 8, 20 nM Ms-LigD and 20 nM Ms-Ku. (B) Ms-LigD and Ms-PolDom super shifted both the U- and Cd-Ku’s and the U-Ku, respectively. Reaction mixtures contained 10 nM labeled dsDNA 33 bp duplex. Lane 1, labeled probe only; lane 2, 0.8 mM U-Ku; lane 3, 0.8 mM Cd-Ku; lane 4, 0.5 mM Ms-PolDom; lanes 5–7, 0.2, 0.4, and 0.8 mM U-Ku in addition to Ms-PolDom (0.5 mM); lane 8, 0.5 mM Ms-LigD; lanes 9–11, 0.2, 0.4, and 0.8 mM U-Ku in addition to Ms-LigD (0.5 mM); and lanes 12–14, 0.2, 0.4, and 0.8 mM Cd-Ku in addition to Ms-LigD (0.5 mM).
exploited yeast-based assays previously used to document the functional link between Mt-Ku and Mt-LigD (Della et al., 2004). Various combinations of phage and bacterial Ku and LigD were expressed in Saccharomyces cerevisiae made NHEJ deficient by host yku70 (Ku) and dnl4 (ligase IV) mutations. We first transformed these yeasts with plasmids linearized in the MET15 marker gene by restriction enzymes that generated fully compatible 4 base overhangs with 50 or 30 polarity. Transformation to Met+ by these plasmids, which requires repair of the DSB by NHEJ, was observed above background levels only when both ectopic Ku and ligase
Bacterial and phage Ku and LigD proteins were expressed in dnl4 yku70 yeast strains lacking endogenous NHEJ in the indicated combinations, and NHEJ efficiency was examined by plasmid recircularization and chromosomal suicide deletion assays as described in the Experimental Procedures. (A) Plasmid DSBs were created with either NsiI (4 base 30 overhangs, top) or NcoI (4 base 50 overhangs, bottom). NHEJ capacity is expressed as the Met+ transformation efficiency by these plasmids normalized to the His+ transformation efficiency by a cotransformed circular HIS3-marked plasmid. (B) Chromosomal DSBs in ADE2 were created by expressing the mega-endonuclease I-SceI (4 base 30 overhangs). NHEJ capacity is expressed as the fraction of cells that survived I-SceI induction as Ade+ (w1% for wild-type yeast). Results are plotted on a log scale. Each bar represents the mean 6 standard deviation (error bar) from three independent experiments. For each assay, U-Ku stimulated LigD-dependent NHEJ, although to different extents in plasmid and chromosomal assays as compared to Mt-Ku and Ms-Ku.
were expressed (Figure 3A), demonstrating that the proteins must act together. U-Ku and the bacterial Ku’s could each stimulate joining by both Mt- and Ms-LigD (Figure 3A), demonstrating that the phage and bacterial Ku’s were redundant and promiscuous in their ability to recruit LigD across bacterial species boundaries. We also employed an assay in which a chromosomal DSB is made in vivo in the ADE2 marker by the endonuclease I-SceI (Della et al., 2004; Karathanasis and Wilson, 2002), where only NHEJ can accurately repair the DSB to Ade+. Repair by LigD was again facilitated by U-Ku, although here the effect was not as pronounced as with Ms-Ku and Mt-Ku (Figure 3B). These results establish that, like the bacterial Ku proteins, U-Ku can function with LigD in NHEJ of single DSBs in the absence of other phage or bacterial proteins.
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Mycobacteriophage Omega and Corndog Utilize NHEJ to Facilitate Genome Circularization The ability of U- and Cd-Ku to facilitate LigD-dependent NHEJ predicted a role for NHEJ in the bacteriophage life cycle that, given that such a role was previously undocumented, in turn predicted that Omega and Corndog must have atypical features. Lambda-like dsDNA bacteriophage typically enter the cell as linear genomes, which are promptly circularized by self-association of long (R9 bases) complementary overhangs called cohesive (cos) ends (Ellis and Dean, 1985). We recently described the profile of rejoining of overhangs of varying length in yeast and showed that this length is a critical parameter in determining the repair mechanism, with repair becoming strictly Ku and NHEJ dependent when overhangs became shorter than six bases (Daley and Wilson, 2005). As genome circularization was a strong candidate for a phage Ku function, we hypothesized that Omega and Corndog would have short cos overhangs. Indeed, comparison of sequences obtained directly from the linear genome with sequences obtained from the genome circularized by ligation confirmed that both Omega and Corndog have 4 base 30 complementary overhanging ends (Figure S6). In contrast, other lambda-like mycobacteriophage described in the same studies as Corndog and Omega (Pedulla et al., 2003), but lacking a phage Ku homolog, showed more typical nine or ten base overhangs expected to show a favorable association equilibrium (Ellis and Dean, 1985). Only circular genomes can support rolling circle replication used to make progeny phage and maintain infectivity. Abolition of NHEJ should therefore lead to lost infectivity by Corndog and Omega, if the circularization hypothesis is correct. To test this, we utilized M. smegmatis strains deficient in Ku (DKu), LigD (DLigD), and a double-knockout strain (DKu, DLigD) (Korycka-Machala et al., 2006). Infection of wild-type M. smegmatis with either Omega or Corndog phage resulted in significant plaque formation on plates (Figure 4A). A similar level of infection was also observed in the DKu strain, consistent with the interpretation that the phage Ku is able to functionally replace the host Ku in phage genome NHEJ, as was observed in the yeast plasmid experiments. However, in a DLigD strain, as well as in a DKu, DLigD strain, absolutely no plaques were formed (Figure 4A), demonstrating that host LigD is essential for phage propagation. To confirm that this phenotype was due to LigD itself, we reintroduced Ms-LigD on an acetamide-inducible plasmid. This genetic complementation resulted in a complete restoration of plaque formation by both Omega and Corndog in the DLigD strain (Figure 4B). Importantly, LigD is a multifunctional protein, but our model predicts that the ligation activity of LigD will be specifically essential for plaque production. To test this, we mutated Ms-LigD gene at two regions (Figure S7), replacing the essential ligase active site lysine with alanine (K484A; ligase domain) and substituting conserved polymerase aspartate residues with alanine (D136A, D138A; PolDom). Although M. smegmatis DLigD strains, transformed with plasmids carrying either the wild-type Ms-LigD or Ms-LigD (D136A, D138), supported infection and plaque formation by Omega and Corndog, there was a clear absence of plaques in strains
Figure 4. Ms-LigD, but Not Ms-Ku, Is Required for Omega and Corndog Plaque Formation in M. smegmatis (A) Plaques formed when either wild-type or DKu M. smegmatis strains were infected with Omega or Corndog phage lysate. However, no plaques formed when either DLigD or DLigD, DKu strains were infected with Omega or Corndog phage lysate. (B) The DNA ligase, but not the polymerase, activity of Ms-LigD was required for plaque formation. Plaques appeared when the LigD deletion strain is complemented with Ms-LigD expressed from an acetamide-inducible plasmid. Plaques also formed when the LigD deletion strain was complemented with Ms-LigD (containing catalytic mutants that prevent polymerase activity) were infected with either phage lysate. However, plaques did not appear when a LigD deletion strain complemented with Ms-LigD (containing a catalytic mutation [K484A] that prevented ligase activity) was infected with either phage lysate.
carrying plasmid pLigD (K484A) (Figure 4B). Thus, the ligation activity of the host NHEJ ligase is indeed essential for the life cycle of these mycobacteriophage. In contrast, the polymerase activity of Ms-LigD is dispensable (Figure 4B), presumably because the genome ends are generated by the observed Corndog and Omega terminase (Pedulla et al., 2003). Terminase-generated ends are expected to be uniformly complementary and require no processing prior to ligation, as typical for all bacteriophage. A corollary is that the NHEJ process that circularizes Corndog and Omega must be predominantly accurate, without nucleotide additions and deletions, so that subsequent cleavage of the cos region by terminase during rolling circle replication can proceed and create the uniform ends observed in the genome (Figure S6). This is seemingly distinct from plasmid transformation studies of M. smegmatis, which have suggested that its NHEJ is highly imprecise (Gong et al., 2005). PCR analysis of phage genomic DNA extracted from bacteria infected with either Omega or
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Corndog phage confirmed the presence of closed-circular viral genomic DNA species in wild-type M. smegmatis. (Figure S8). Finally, our model predicts that infection by phage with longer overhangs should be NHEJ, and therefore LigD, independent. Indeed, mycobacteriophage D29 (Pedulla et al., 2003), which contains a nine base overhanginfected M. smegmatis (DLigD) as readily as the wildtype strain (Figure S9), additionally demonstrating that this strain is not deficient in supporting phage replication when NHEJ-dependent circularization is not required. Conclusions We have presented data demonstrating that selected mycobacteriophage of the lambda family depend on the host-encoded NHEJ ligase for circularization of their dsDNA genomes, in a manner that correlates precisely with the shortness of their cos overhangs. LigD’s essential function is ligation, as evidenced by selective point mutants. The host Ku is not required apparently because these bacteriophage, Corndog and Omega, encode their own Ku homolog, which interacts functionally with the host LigD in vitro and can activate it for NHEJ in ectopic NHEJ reconstitution in vivo. In addition to facilitating ligation, bacteriophage Ku may also serve to specifically protect the phage genomic ends from degradation upon cellular entry (Figure S5), providing an explanation for the existence of a phage-encoded Ku’s as opposed to sequestration of the entire cellular NHEJ machinery (Bowater and Doherty, 2006; d’Adda di Fagagna et al., 2003; Pitcher et al., 2005b). Taken together, our results emphasize that circularization of dsDNA genomes of viruses, and indeed all ‘‘repair’’ steps of viral life cycles, are subject to the same rules and conditions as chromosomal DSBs, and provide a framework for understanding the importance of DSB repair processes in viral infection. For example, the dsDNA genome of herpes simplex virus type 1 (HSV-1) can be circularized (Mocarski and Roizman, 1982), although it is uncertain whether this is a required step in lytic infection (Strang and Stow, 2005). Our results point to the possibility that such circularization events may indeed be a normal part of the viral life cycle and that NHEJ might be expected to play an important role in this process given its predominance in eukaryotic cells. Interestingly, the HSV protein vmw110 induces the degradation of the catalytic subunit of DNA-dependent protein kinase (Parkinson et al., 1999), but the effect of this degradation on the balance of apoptotic signaling and repair of viral DSB ends is not established. In a different viral type, HIV genomes can be circularized after reverse transcription into dsDNA by at least two mechanisms that preserve one or both of the long terminal repeats (LTRs) (Jeanson et al., 2002; Kilzer et al., 2003; Li et al., 2001; Skalka and Katz, 2005). The fact that Ku deficiency impairs only circularization that preserves both LTRs suggests that this represents NHEJ, whereas one-LTR circularization may perhaps be analogous to single-strand annealing (Ivanov et al., 1996). Such processes may have substantial indirect effects on the ability of a cell to support infection, for example by leading to persistence of DSB ends or altered damage signaling, in contrast to the suggested direct role of NHEJ in retroviral integration (Skalka and Katz, 2005).
Experimental Procedures Expression Constructs Full-length sequences for Ms-LigD and Ms-Ku were amplified by PCR from M. smegmatis mc2155 genomic DNA. Omega-Ku (gp206) and Corndog-Ku (gp87) were amplified from phage genomic DNA. All PCR products were cloned into pET28a (Novagen). Ms-PolDom was constructed by replacing R300 (Ms-LigD numbering) with a stop codon using pMs-LigD as a template. Overexpression and Purification of Constructs Ms-PolDom and Ms-LigD were overexpressed and purified as described previously (Gong et al., 2005; Pitcher et al., 2005a). Recombinant Ku proteins (Ms-Ku, Omega-Ku, and Corndog-Ku) were purified as described previously (Weller et al., 2002). EMSAs EMSAs were conducted by using linear DNA duplexes as described previously (Weller et al., 2002). Plasmid Repair Assay Plasmid repair assays were performed as previously described (Della et al., 2004). One-hundred nanograms of PvuI-cut pUC18 and 0–125 nM U- or Cd-Ku, and when present Ms-Ku (20 nM), were incubated for 15 min. Ms-LigD (20 nM) was then added, and the reactions were incubated for 1 hr at 37 C. The reactions were examined by PCR, using forward and reverse primers upstream and downstream respectively of the restriction site. PCR products were separated on a 1% (w/v) agarose gel stained with ethidium bromide. Yeast NHEJ Assays Plasmid-based expression of Mt-Ku and Mt-LigD in yeast from the ADH1 promoter as Myc-NLS fusion proteins was as previously described (Della et al., 2004). Ms-Ku, Ms-LigD, and U-Ku were expressed similarly in various combinations. The yeast strain was YW1231 (Della et al., 2004), which was dnl4 yku70 mutant to make it NHEJ deficient, and bore the I-SceI suicide deletion allele for monitoring chromosomal repair (Karathanasis and Wilson, 2002). For plasmid assays, 100 ng of pRS411 cut with either NcoI or NsiI within its MET15 marker was cotransformed with 20 ng HIS3-marked pRS413 (Brachmann et al., 1998) as previously described (Daley and Wilson, 2005). Repair efficiency is expressed as the ratio of Met+ to His+ colonies. For chromosomal assays, yeast were plated to galactose medium to induce I-SceI expression. Repair efficiency is expressed as the ratio of Ade+ colonies on galactose, indicating repair by NHEJ, to total viable colonies on parallel glucose plates. Growth and Infection of Mutant M. smegmatis Strains Wild-type M. smegmatis and mutant strains were grown from single colonies at 37 C with shaking in Middlebrook 7H9 broth (Difco) supplemented with 7H9 ADC enrichment media (Difco) to an OD600 of w1. Complementation of DLigD strains required 0.2% acetamide in culture media to stimulate transcription of the LigD gene on the plasmid. If necessary, the ODs were adjusted, before 0.1 ml of each culture was mixed with 0.1 ml of tenfold serial dilutions (in SM buffer) of phage lysate and incubated at 37 C for 20 min. Then, 150 ml of this mixture was added to 7 ml of top agar (diluted 7H10 Middlebrook agar [Difco] supplemented with OADC enrichment media [Difco]) quickly mixed and plated out onto 7H10 Middlebrook agar supplemented with OADC and incubated at 37 C for 24–48 hr. To confirm that circularization was part of the phage life cycle, primer pairs were designed for the 50 and 30 termini of either the Omega or Corndog genome that would produce a 500 bp fragment by PCR if the genomes were indeed circularized during plaque formation. Primers that would produce a 1 Kb fragment whether the genomes were linear or circular were also used as controls. Directed Mutagenesis of Ms-LigD Gene The key amino acids of the polymerase (136D, 138D) and ligase (484K) active sites of LigD were replaced with alanine (A) by using PCR-directed mutagenesis. The resultant PCR products were cloned into pGemT-easy vector and sequenced to confirm the introduced mutation (Figure S7). Finally, the mutated ligD genes were
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introduced into the pJam2 shuttle vector under the control of an acetamide promoter (Triccas et al., 1998). The resultant vectors pJam2-ligDmutL (ligase mutant) and pJam2-ligDmutP (polymerase mutant) were introduced into an M. smegmatis strain carrying an unmarked deletion in the wild-type ligD gene (M. smegmatis DligD) (Korycka-Machala et al., 2006). Supplemental Data Supplemental Data include nine figures and can be found with this article online at http://www.molecule.org/cgi/content/full/23/5/ 743/DC1/. Acknowledgments We thank N. Brissett, R. Curtis, E. Davis, M. Pedulla, C. Peebles, and J. van Kessel for their valuable assistance and comments. A.J.D. is a Royal Society University Research Fellow and A.J.D.’s laboratory is supported by grants from the Biotechnology and Biological Sciences Research Council, American Institute for Cancer Research, and Cancer Research-UK. T.E.W. is supported by grants from United States Public Health Service and the Pew Charitable Trusts. Received: April 25, 2006 Revised: June 21, 2006 Accepted: July 10, 2006 Published: August 31, 2006 References Bowater, R., and Doherty, A.J. (2006). Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining. PLOS Genet. 2, 93–99. 10.1371/journal.pgen.0020008. Bliss, T.M., and Lane, D.P. (1997). Ku selectively transfers between DNA molecules with homologous ends. J. Biol. Chem. 272, 5765– 5773. Brachmann, C.B., Davies, A., Cost, G.J., Caputo, E., Li, J., Hieter, P., and Boeke, J.D. (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132. d’Adda di Fagagna, F., Weller, G.R., Doherty, A.J., and Jackson, S.P. (2003). The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku. EMBO Rep. 1, 47–53. Daley, J.M., and Wilson, T.E. (2005). Rejoining of DNA double-strand breaks as a function of overhang length. Mol. Cell. Biol. 25, 896–906. Daniel, R., Katz, R.A., and Skalka, A.M. (1999). A role for DNA-PK in retroviral DNA integration. Science 284, 644–647. Daniel, R., Greger, J.G., Katz, R.A., Taganov, K.D., Wu, X., Kappes, J.C., and Skalka, A.M. (2004). Evidence that stable retroviral transduction and cell survival following DNA integration depend on components of the nonhomologous end joining repair pathway. J. Virol. 78, 8573–8581. Della, M., Palmbos, P.L., Tseng, H.M., Tonkin, L.M., Daley, J.M., Topper, L.M., Pitcher, R.S., Tomkinson, A.E., Wilson, T.E., and Doherty, A.J. (2004). Mycobacterial Ku and ligase proteins constitute a two-component NHEJ repair machine. Science 306, 683–685. Doherty, A.J., and Jackson, S.P. (2002). How Ku makes ends meet. Curr. Biol. 11, R920–R924. Downs, J.A., and Jackson, S.P. (1999). Involvement of DNA endbinding protein Ku in Ty element retrotransposition. Mol. Cell. Biol. 19, 6260–6268. Ellis, D.M., and Dean, D.H. (1985). Nucleotide sequence of the cohesive single-stranded ends of Bacillus subtilis temperate bacteriophage phi 105. J. Virol. 55, 513–515. Gong, C., Martins, A., Bongiorno, P., Glickman, M., and Shuman, S. (2004). Biochemical and genetic analysis of the four DNA ligases of mycobacteria. J. Biol. Chem. 279, 20594–20606. Gong, C., Bongiorno, P., Martins, A., Stephanou, N.C., Zhu, H., Shuman, S., and Glickman, M.S. (2005). Mechanism of nonhomologous end-joining in mycobacteria: A low-fidelity repair system driven by Ku, ligase D and ligase C. Nat. Struct. Mol. Biol. 12, 304–312.
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