ABSTRACT
Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction. Mutant K27A is unable to form the covalent ligase-(Lys-[epsilon]N-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick. This shows that the active site lysine is not required for the strand closure reaction. K27A binds to nicked DNA-adenylate, but not to a standard DNA nick. This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition. Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation. R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.
The ATP-dependent DNA ligases catalyze the joining of 5'-phosphate-terminated strands to 3'-hydroxyl-terminated strands via three sequential nucleotidyl transfer reactions (1-3). In the first step, attack on the [alpha]-phosphate of ATP by DNA ligase results in displacement of pyrophosphate and formation of a covalent ligase-adenylate intermediate in which AMP is linked to the [epsilon]-amino group of a lysine. The active site lysine residue is located within a conserved motif, KxDGxR (4). The AMP is then transferred to the 5'-monophosphate terminus of a nicked DNA duplex to form the DNA-adenylate intermediate, which consists of an inverted (5')-(5') pyrophosphate bridge structure, AppN. Attack by the 3'-OH-terminated strand of the nicked duplex on DNA-adenylate seals the nick and releases AMP.
We are examining the structure and function of the eukaryotic DNA ligases using virus encoded enzymes as models. The 298 amino acid Chlorella virus PBCV-1 DNA ligase is the smallest eukaryotic DNA ligase known and likely constitutes the minimal catalytic unit (5). Initial studies of strand joining and DNA recognition were performed using purified recombinant protein and duplex DNA substrates containing a single nick. We showed that PBCV-1 ligase forms a stable complex with nicked DNA prior to reaction at the nick and that the enzyme discriminates at the DNA binding step between nicked DNA molecules that can be sealed versus gapped molecules that are not ligated (4). Preferential binding to DNA nicks has also been demonstrated for vaccinia virus DNA ligase and mammalian DNA ligase III (6-8).
In the present study we further examine the basis for DNA recognition by PBCV-1 DNA ligase. We show that binding is indeed nick specific and that properly positioned 5'-phosphate and 3'-hydroxyl termini are required for stable binding. In addition, we have initiated a mutational analysis of PBCV-1 DNA ligase, focusing on the active site motif TPKIDGIR. We find that the conserved lysine, aspartate and arginine residues are essential for overall strand joining. Remarkably, mutations of these residues elicit distinctive effects on each of the three nucleotidyl transfer reactions.
PBCV-1 DNA ligase was expressed in bacteria and purified to homogeneity as described (5).
Ligase substrate
The standard substrate used in ligase assays was a 36 bp nucleic acid duplex containing a centrally placed nick. This DNA was formed by annealing two 18mer oligodeoxyribonucleotides to a complementary 36mer strand (6). The 18mer constituting the 5'-phosphate-terminated strand d(ATTCCGATAGTGACTACA) was 5'-32P-labeled and gel purified as described (6). The labeled 18mer was annealed to the complementary 36mer DNA (the template strand) in the presence of a 3'-OH 18mer strand d(CATATCCGTGTCGCCCTT) as described (9).
Reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 10 mM MgCl2, 1 mM ATP, 5'-32P-labeled nicked duplex substrate and enzyme were incubated at 22°C for 10 min. Reactions were initiated by addition of enzyme and halted by addition of 1 µl 0.5 M EDTA and 5 µl formamide. The samples were heated at 95°C for 5 min and then electrophoresed through a 17% polyacrylamide gel containing 7 M urea in TBE (90 mM Tris-borate, 2.5 mM EDTA). Where indicated the extent of ligation [36mer/(18mer + 36mer)] was determined by scanning the gel using a Fujix BAS1000 phosphorimager.
Binding reaction mixtures (20 µl) containing 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 32P-labeled nucleic acid ligand and DNA ligase as specified were incubated for 10 min at 22°C. Glycerol was added to 5% and the samples were electrophoresed through a 6% polyacrylamide gel in TBE at 70 V for 2 h. Free ligand and ligase-nucleic acid complexes of retarded electrophoretic mobility were visualized by autoradiography of the dried gel.
Missense mutations in the PBCV-1 ligase gene were programed by synthetic oligonucleotides using the two stage PCR-based overlap extension strategy (10). An NdeI-BamHI restriction fragment of each PCR-amplified gene was inserted into pET16b so as to place the coding sequence in-frame with an N-terminal leader sequence encoding 10 tandem histidines. The presence of the desired mutation was confirmed in every case by sequencing the entire ligase insert; the occurrence of PCR-generated mutations outside the targeted region was thereby excluded. The pET-His-ligase plasmids were transformed into Escherichia coli BL21(DE3). Single colonies were inoculated into LB medium containing 0.1 mg/ml ampicillin and grown at 37°C until the A600 reached ~0.8. The cultures (100 ml) were placed on ice for 30 min, then adjusted to 0.4 mM IPTG and subsequently maintained at 17°C for 6 h with continuous shaking. Cells were harvested by centrifugation and pellets were stored at -80°C. All subsequent procedures were performed at 4°C. Cell lysis was achieved by treatment of thawed, resuspended cells with 0.75 mg/ml lysozyme and 0.1% Triton X-100 in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 10% sucrose. Insoluble material was removed by centrifugation at 18 000 r.p.m. for 30 min in a Sorvall SS34 rotor. The supernatants were mixed with 1 ml Ni-NTA-agarose resin (Qiagen) for 1 h. The slurries were poured into a column and then washed with lysis buffer. The columns were eluted stepwise with IMAC buffer (20 mM Tris-HCl, pH 7.9, 50 mM NaCl, 10% glycerol) containing 5, 25, 50, 100, 200 and 500 mM imidazole. The polypeptide composition of the column fractions was monitored by SDS-PAGE. The His-tagged ligases were eluted at 200 mM imidazole. The eluates were applied to 1 ml columns of phosphocellulose that had been equilibrated with 50 mM Tris-HCl, pH 8.0, 10% glycerol. The columns were eluted stepwise with 50, 100, 200, 400 and 500 mM NaCl in 50 mM Tris-HCl, pH 8.0, 10% glycerol. The ligases were recovered in the 0.4 M NaCl fraction. The protein concentrations of the enzyme preparations were determined using the BioRad dye reagent with bovine serum albumin as standard.
We compared the binding of purified recombinant PBCV-1 ligase to a nicked duplex DNA ligand containing either a 5'-phosphate or a 5'-hydroxyl at the nick. The DNAs consisted of a 5'-32P-labeled 3'-OH-terminated hairpin oligonucleotide and an unlabeled 18mer strand (either 5'-phosphate- or 5'-hydroxyl-terminated) annealed to the 5'-tail of the hairpin strand (Fig. 1). PBCV-1 ligase efficiently sealed the substrate containing the 5'-phosphate-terminated 18mer, but was incapable of sealing the molecule containing a 5'-hydroxyl at the nick, even in enzyme excess (data not shown). A native gel mobility shift assay was used to directly examine the binding of PBCV-1 ligase to the nicked hairpin ligands. Binding reactions were performed in the absence of ATP and a divalent cation so as to preclude conversion of substrate to product during the incubation (5). Mixing the ligase with 32P-labeled nicked DNA containing a 5'-phosphate at the nick resulted in formation of a discrete protein-DNA complex that migrated more slowly than the free DNA during electrophoresis through a 6% native polyacrylamide gel (Fig. 1). The yield of this complex was proportional to input ligase. No specific complex was detected when PBCV-1 ligase was incubated with the hairpin strand alone or with a nicked DNA ligand containing a 5'-hydroxyl at the nick (Fig. 1). We also found that PBCV-1 ligase did not form a complex with a sealed hairpin duplex molecule, i.e. the re-isolated product of ligation of the nicked hairpin (Fig. 1). This experiment shows that DNA ligase binds specifically at a nick and is capable of discriminating at the substrate binding step between ligands containing 5'-phosphate versus 5'-hydroxyl moieties at the nick. A 3'-hydroxyl terminus, though required for ligation, is clearly not sufficient for substrate binding.
The active site lysine of every ATP-dependent DNA ligase is situated within a conserved sequence KxDGxR, referred to as motif I (4,11,12). Motif I is one of six protein segments that are conserved with the same order and with similar spacing among the ATP-dependent ligases and the GTP-dependent mRNA capping enzymes (12-14). The crystal structures of T7 DNA ligase with bound ATP and the PBCV-1 RNA capping enzyme with bound GTP show that the lysine and arginine sidechains make contact with the nucleotide (15,16).
The sequence surrounding the presumptive active site residue (Lys27) of PBCV-1 DNA ligase is TPKIDGIR. In order to examine the function of motif I, we introduced alanine substitutions for Lys27, Asp29, Gly30 and Arg32. We also mutated Thr25; this was done in the light of the finding that the residue located two amino acids upstream of the active site lysine of T7 DNA ligase (which is a glutamate in the T7 enzyme) contacts the adenine base of ATP (15). In addition to the alanine substitutions, we engineered conservative mutations K27R, D29E, D29N and R32K. The wild-type PBCV-1 ligase and the eight mutant ligases were expressed in bacteria as N-terminal His-tagged fusion proteins, then partially purified from soluble lysates by Ni-agarose and phosphocellulose column chromatography. The enzyme preparations were highly enriched with respect to the 34 kDa PBCV-1 ligase polypeptide (Fig. 2A).
Mutational effects on ligation were quantitated by enzyme titration (Fig. 3A). The extent of ligation was proportional to input enzyme for the wild-type ligase and for each of the catalytically active mutants. All reactions saturated with ~80-85% of the 32P-labeled 18mer stand converted to 36mer in 10 min (Fig. 3A and data not shown). This upper limit of ligation probably reflected incomplete annealing of all three component strands to form the nicked substrate. The specific activities of the mutants were calculated in the linear range of enzyme dependence and then normalized to the wild-type specific activity. The values were as follows: wild-type, 100%; T25A, 64%; K27R, 3%; K27A, <0.01%; D29E, 27%; D29N, 4%; D29A, <0.01%; G30A, 3%; R32K, 8%; R32A, <0.01%. Activities of the defective mutants K27A, D29A and R32A are upper limit estimates based on titrating enzyme from 34 to 270 ng input protein (not shown). We conclude from the alanine scanning results that Thr25 is non-essential for strand joining, whereas conserved residues Lys27, Asp29, Gly30 and Arg32 are critical for ligation.
We performed a kinetic analysis of strand joining by wild-type PBCV-1 ligase and the T25A, K27R, D29E, D29N, G30A and R32K mutants. Ligase was added in ~5-fold molar excess over the nicked DNA substrate in order to approximate single turnover conditions. The reactions proceeded to similar end-points, with ~75-90% of the 32P-labeled 18mer strand converted to 36mer (Fig. 3B and data not shown). However, the rates of approach to the end-point differed. The apparent ligation rate constants were calculated by fitting the data to a single exponential (9). The values for kobs were as follows: wild-type, 0.23 s-1/ (100%); T25A, 0.15 s-1/ (67%); K27R, 0.034 s-1/ (15%); D29E, 0.034 s-1/ (15%); D29N, 0.005 s-1/ (2%); G30A, 0.031 s-1/ (14%); R32K, 0.48 s-1/ (20%).
Elimination of the functional groups of Lys27, Asp29 and Arg32 by alanine substitution abrogated strand joining by PBCV-1 DNA ligase. To gain insights into how these essential residues contribute to the overall ligation reaction we examined the effects of the K27A, D29A and R32A mutations on each of the three components steps. The initial step in DNA ligation involves formation of a covalent enzyme-adenylate intermediate, EpA. Formation of EpA by PBCV-1 ligase can be detected by label transfer from [[alpha]-32P]ATP to the enzyme (5). Incubation of wild-type ligase in the presence of [[alpha]-32P]ATP and a divalent cation resulted in formation of a nucleotidyl-protein adduct that migrated as a single species during SDS-PAGE (Fig. 4). The K27A mutant was inert in enzyme-adenylate formation, as expected. R32A also formed no detectable EpA. However, the D29A mutant did react with ATP to from the covalent intermediate. This suggests that Asp29 is essential for a step subsequent to EpA formation.
The second step of ligation is the transfer of AMP from ligase-adenylate to the 5'-phosphate terminus at the nick to form DNA-adenylate. This intermediate is not detected during ligation of nicked DNA by wild-type PBCV-1 ligase (5). However, DNA-adenylate accumulates under reaction conditions that drastically slow the third step of the ligation reaction, attack of the 3'-OH acceptor strand on DNA-adenylate (6). This can be achieved by introducing a 1 nt gap between the reactive 3'-hydroxyl and 5'-phosphate DNA strands (Fig. 5). Reaction of the gapped substrate with stoichiometric levels of wild-type PBCV-1 ligase in the presence of ATP and magnesium resulted in conversion of the 5'-32P-labeled 18mer strand into an adenylated species (AppDNA) that migrated ~1 nt slower than the input 18mer during polyacrylamide gel electrophoresis (Fig. 5). DNA-adenylate was more abundant that the 36mer ligation product. The K27A and R32A mutants were incapable of DNA adenylation. This can be ascribed simply to the inability of these two proteins to form enzyme-adenylate. The D29A mutant was also incapable of forming DNA-adenylate under the same conditions (Fig. 5), even though this protein was active in enzyme-adenylate formation (Fig. 4). We surmise that Asp29 is required for step 2 of the ligation reaction.
Step 3 of the ligation reaction was assayed by the ability of the wild-type and mutant enzymes to seal a pre-adenylated nicked duplex DNA (Fig. 6). The adenylated strand used to form this substrate was synthesized by ligase-mediated AMP transfer to the 5'-32P-labeled strand of a DNA molecule containing a 1 nt gap. The radiolabeled AppDNA strand was gel purified and annealed to an unlabeled 42 nt hairpin strand to form the structure shown in Figure 6. This substrate was reacted with ligase in the presence of magnesium. The wild-type PBCV-1 ligase generated a 60mer ligation product. The active site mutant K27A also catalyzed the strand joining step at a pre-adenylated nick, even though K27A was completely inactive in catalyzing the first two partial reactions. Indeed, more ligated product was formed by K27A than by wild-type ligase (Fig. 6). We conclude that the lysine nucleophile is dispensable for phosphodiester bond formation. The R32A mutant, on the other hand, was inactive in sealing a pre-adenylated nick. Hence, the defect of this mutant could not be bypassed by eliminating the requirement for the adenylyl transferase reaction. We surmise that Arg32 is essential for step 3 as well as for step 1. The D29A mutant was capable of closing a pre-adenylated nick, although the extent of reaction was lower than that of wild-type ligase (Fig. 6). We infer that a block to step 2 is the principal cause of the catalytic defect of D29A.
The D29A mutant protein bound to the nicked hairpin DNA substrate to yield a single protein-DNA complex that was indistinguishable in its electrophoretic mobility from the protein-DNA complex formed by the wild-type ligase (Fig. 7 and data not shown). In contrast, neither K27A nor R32A were capable of forming a gel-shifted complex on the nicked substrate (Fig. 7). These results underscore a correlation between defective nick recognition and inability to form the ligase-AMP complex.
The recombinant wild-type PBCV-1 DNA ligase purified from bacteria contains a significant fraction of ligase-adenylate (5). In contrast, the purified active site mutant K27A is exclusively in the unadenylated form. We compared the ability of the wild-type and K27A proteins to bind to 32P-labeled nicked duplex DNA and nicked DNA-adenylate molecules (Fig. 8). The wild-type ligase bound to the nicked DNA, but the K27A mutant did not. We would attribute this to the absence of a bound adenylate moiety on the enzyme. If occupancy of the adenylate binding site is important for substrate recognition, then we would predict that K27A should bind to a nicked ligand containing an adenylated DNA strand at the nick. This was indeed the case (Fig. 8). Note that the wild-type ligase bound less well to DNA-adenylate than to nicked DNA. This might be expected, given that most of the wild-type ligase molecules contain a covalently bound AMP molecule that would sterically hinder binding to the adenylated DNA. We presume that the observed binding by the wild-type enzyme to AppDNA is mediated by the non-adenylated fraction of the enzyme preparation.
The results presented above enhance our understanding of substrate recognition and catalysis by ATP-dependent DNA ligases in the following respects: (i) PBCV-1 ligase is shown to bind specifically to a 3'-hydroxyl/5'-phosphate nick in duplex DNA; (ii) mutational analysis illuminates essential roles for the conserved lysine, aspartate and arginine residues of motif I at different steps of the ligase reaction; (iii) the binding properties of the active site lysine mutant suggest that either covalent or non-covalent occupancy of the AMP binding site on the enzyme is important for nick recognition.
PBCV-1 DNA ligase binds to a nicked DNA duplex containing reactive 3'-OH and 5'-PO4 termini. It does not bind to a continuous DNA duplex, to a tailed duplex or even to a nicked ligand containing non-ligatable 3'-OH and 5'-OH termini. The enzyme also discriminates nicks from gaps (5). Insofar as the PBCV-1 enzyme can be construed as the minimal functional unit of an ATP-dependent DNA ligase, we surmise that structural elements within the core catalytic domain are sufficient to mediate nick recognition.
We conclude from the alanine scanning results that Thr25 is non-essential for strand joining, whereas conserved residues Lys27, Asp29, Gly30 and Arg32 are critical for nick ligation. The lack of mutational effect at Thr25 is pertinent in the light of the crystal structure of T7 DNA ligase, in which a glutamate sidechain located two residues upstream of the active site lysine contacts the 6-amino group of the adenine ring of ATP (15). It has been suggested that the contacts made by this residue of the T7 enzyme may explain the specificity of the ligase for ATP (15). Most, but not all, members of the ATP-dependent DNA ligase family have acidic sidechains at this position. Exceptions include the ligases of bacteriophage T4 (glutamine), African swine fever virus (histidine) and PBCV-1 (threonine). Any of these residues could make polar contacts with N6 of adenine. The fact that Thr25 can be replaced by alanine with little effect implies that either: (i) any contacts make by this residue with ATP are not functionally critical; (ii) a residue other than Thr25 in the PBCV-1 ligase makes the adenine-specific contacts invoked for Glu32 of the T7 enzyme. To our knowledge the effects of mutating Glu32 of the T7 DNA ligase have not been reported.
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Chlorella virus DNA ligase
Ligation assay
Nucleic acid binding assay
Ligase mutants
Results
Binding of PBCV-1 ligase to nicked DNA requires a 5'-phosphate moiety at the nick
Mutational analysis of the ligase active site
Mutational effects on strand joining activity
Single turnover ligation
Enzyme-adenylate formation by K27A, D29A and R32A
DNA-adenylate formation
Phosphodiester bond formation at a pre-adenylated nick
DNA binding by K27A, D29A and R32A
Nick recognition requires occupancy of the adenylate binding site on the enzyme
Discussion
Nick recognition
Catalytic roles of amino acids in motif I
References
REFERENCES
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