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Nucleic Acids Research Pages 3536-3541  

Specificity and fidelity of strand joining by Chlorella virus DNA ligase
Introduction
Materials And Methods
   Ligase substrate
   Ligation assay
   Nucleic acid binding assay
Results
   Ligation of DNA strands on a DNA versus RNA template
   Ligation of RNA strands on a DNA template
   Ligation of a DNA substrate containing a 5[prime]-ribonucleotide at the nick
   Effects of base mismatches at the reactive 5[prime]-phosphate and 3[prime]-hydroxyl positions
Discussion
   Discrimination of RNA versus DNA strands
   Embedding of single ribonucleotides in DNA
   Ligase fidelity
References

Specificity and fidelity of strand joining by Chlorella virus DNA ligase

Specificity and fidelity of strand joining by Chlorella virus DNA ligase

Verl Sriskanda, Stewart Shuman*

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA

Received April 8, 1998; Revised and Accepted June 4, 1998

ABSTRACT

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates consisting of a 5[prime]-phosphate-terminated strand and a 3[prime]-hydroxyl-terminated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template. Whereas PBCV-1 ligase efficiently joins a 3[prime]-OH RNA to a 5[prime]-phosphate DNA, it is unable to join a 3[prime]-OH DNA to a 5[prime]-phosphate RNA. The ligase discriminates at the substrate binding step between nicked duplexes containing 5[prime]-phosphate DNA versus 5[prime]-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5[prime]-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide on the 5[prime]-phosphate side of the nick. Single base mismatches at the nick exert disparate effects on DNA ligation efficiency. PBCV-1 ligase tolerates mismatches involving the 5[prime]-phosphate nucleotide, with the exception of 5[prime]-A:G and 5[prime]-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3[prime]-OH nucleotide include 3[prime]-G:A, 3[prime]-G:T, 3[prime]-T:T, 3[prime]-A:G, 3[prime]-G:G, 3[prime]-A:C and 3[prime]-C:C. Our findings indicate that Chlorella virus DNA ligase has the potential to affect genome integrity by embedding ribonucleotides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations.

INTRODUCTION

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 (1). PBCV-1 ligase resembles other ATP-dependent DNA ligases that catalyze the joining of 5[prime]-phosphate-terminated strands to 3[prime]-hydroxyl-terminated strands via three sequential reactions (2-4). 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 [epsis]-amino group of a lysine. The active site lysine residue is located within a conserved motif, KxDGxR (5-10). The AMP is then transferred to the 5[prime]-monophosphate terminus of a nicked DNA duplex to form the DNA-adenylate intermediate, which consists of an inverted (5[prime])-(5[prime]) pyrophosphate bridge structure, AppN. Attack by the 3[prime]-OH on DNA-adenylate seals the nick and releases AMP.

The Chlorella virus ligase catalyzes efficient strand joining on nicked DNA, but is unable to seal strands across a 2 nt gap (1). Action at a 1 nt gap results in accumulation of high levels of the normally undetectable DNA-adenylate reaction intermediate and relatively little strand joining (1,9). Native gel mobility shift assays show that PBCV-1 ligase forms a stable complex with nicked DNA, but not with any of the following ligands: (i) DNA containing a 1 or 2 nt gap; (ii) the fully sealed duplex product of the ligation reaction; (iii) a singly nicked duplex containing a 5[prime]-OH terminus at the nick (1,9). Properly positioned 5[prime]-phosphate and 3[prime]-OH termini are apparently required for substrate recognition by PBCV-1 ligase. Nick recognition also depends on occupancy of the AMP binding pocket on the enzyme, i.e. active site mutations that abolish the capacity to form the ligase-adenylate intermediate also eliminate nick recognition, whereas a mutation that preserves ligase-adenylate formation but inactivates downstream steps of the strand joining reaction has no effect on binding to nicked DNA (9).

We presume that the ligase interacts with the DNA duplex at sites other than the 5[prime]-phosphate and 3[prime]-hydroxyl moieties, but the nature of such contacts has not been defined. One way to approach this issue is to alter the structure of the nucleic acid substrate without perturbing the 5[prime]-phosphate and 3[prime]-hydroxyl groups at the nick. In the present study, we analyze the ability of PBCV-1 DNA ligase to seal: (i) nicked substrates containing one or more RNA strands; (ii) a nicked substrate containing a single ribonucleotide at the 5[prime]-phosphate position; (iii) substrates containing a single base mismatch at the nick. We find that the Chlorella virus enzyme catalyzes efficient joining of 3[prime]-OH RNA to 5[prime]-phosphate DNA, but not 3[prime]-OH DNA to 5[prime]-phosphate RNA. The inability to seal a 5[prime]-phosphate RNA strand is not attributable to interference by a ribonucleotide at the reactive 5[prime]-phosphate terminus. Strand joining is inhibited by certain base mismatches at the nick.

MATERIALS AND METHODS

Ligase substrate

The standard substrate used in ligase assays was a 36 bp nucleic acid duplex containing a centrally placed nick. This molecule was formed by annealing two 18mer oligonucleotides to a complementary 36mer strand (11). The 18mer constituting the 5[prime]-monophosphate-terminated strand [either DNA 18mer d(ATTCCGATAGTGACTACA) or RNA 18mer r(AUUCCGAUAGUGACUACA)] was 5[prime]-32P-labeled and gel purified as described (11). The labeled 18mer was annealed to the complementary 36mer DNA (the template strand) in the presence of a 3[prime]-OH 18mer strand [either DNA oligonucleotide d(CATATCCGTGTCGCCCTT) or RNA oligo-nucleotide r(CAUAUCCGUGUCGCCCUU)] as described (12).

Ligation assay

PBCV-1 DNA ligase was expressed in bacteria and purified as described (1). Ligation reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 10 mM MgCl2 or MnCl2 as specified, 1 mM ATP, 5[prime]-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 analyzed by electrophoresis 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 was determined by scanning the gel with a Fujix BAS1000 phosphorimager.

Nucleic acid binding assay

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% (v/v) and the samples were analyzed by electrophoresis through a non-denaturing 6% polyacrylamide gel in TBE at 70 V for 2 h. Free ligand and ligase-nucleic acid complexes of retarded mobility were visualized by autoradiography of the dried gel.

RESULTS

Ligation of DNA strands on a DNA versus RNA template

We compared the ability of PBCV-1 DNA ligase to join two 18mer DNA strands that were annealed to a 20 nt template strand composed of either DNA or RNA. The annealed substrates contained a single nick flanked on both sides by 10 bp of duplex nucleic acid (Fig. 1). Activity was measured by conversion of the 5[prime]-32P-labeled 18mer strand into an internally labeled 36mer product. PBCV-1 DNA ligase catalyzed efficient strand joining when the template strand was composed of DNA; 96% of the 32P-labeled 18mer DNA was ligated in 10 min. In contrast, <0.1% of the 32P-labeled 18mer DNA was ligated when the template strand was RNA (Fig. 1). The substrate specificity of enzymes involved in nucleic acid metabolism can sometimes be relaxed by substituting manganese for magnesium as the divalent cation cofactor. In the case of PBCV-1 DNA ligase, substitution of 10 mM manganese for 10 mM magnesium did not enhance ligation of DNA strands on an RNA template (Fig. 1). Other concentrations of manganese were not tested. (Prior studies had shown that DNA strand joining by PBCV-1 ligase was optimal at 10-20 mM of either divalent cation; 1.)


Figure 1. Ligation of DNA molecules annealed to a RNA template strand. The structure of the nicked duplex substrate used in the ligation reactions is shown; the 32P-labeled 5[prime]-phosphate at the nick is indicated by the dot. The 3[prime]-OH and 5[prime]-phosphate 18mer strands were both DNA; the 20mer template strand was either DNA or RNA. Reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 1 mM ATP, either 10 mM MgCl2 or 10 mM MnCl2, 500 fmol nicked substrate containing a DNA template strand or RNA template strand as indicated and 34 ng DNA ligase were incubated for 10 min at 22°C. The products were resolved by polyacrylamide gel electrophoresis. The positions of the 32P-labeled 18mer substrate and the 36mer ligation product are indicated on the left.

Ligation of RNA strands on a DNA template

Assays of RNA strand ligation were performed using a 36mer duplex substrate containing a centrally positioned nick in which the 5[prime]-phosphate strand, the 3[prime]-OH strand or both strands were composed of RNA. In each case, the 5[prime]-phosphate and 3[prime]-OH strands were annealed to a complementary DNA template strand (Fig. 2A). PBCV-1 DNA ligase readily joined a 3[prime]-OH RNA to a 5[prime]-phosphate DNA to yield a 36mer RNApDNA product with either 10 mM magnesium or manganese as the divalent cation cofactor (Fig. 2B). The enzyme did not join a 3[prime]-OH DNA to a 5[prime]-phosphate RNA to produce a DNApRNA molecule, nor did it join two RNA strands to produce RNApRNA product (Fig. 2B). Substituting manganese for magnesium did not significantly enhance the utilization by PBCV-1 ligase of substrates containing RNA on the 5[prime]-phosphate side of the nick (Fig. 2B). It was noted previously that 10 mM manganese did stimulate RNA strand joining by vaccinia DNA ligase (12).


Figure 2. Ligation of RNA-containing substrates. (A) The structure of the nicked duplex substrate used in the ligation reactions is shown. The 32P-labeled 5[prime]-phosphate at the nick is indicated by the dot. The 3[prime]-OH and 5[prime]-phosphate 18mer strands were either DNA or RNA; the 36mer bridging strand was all-DNA. (B) Ligation reaction mixtures were constituted as described in Figure 1 and contained 500 fmol of the indicated substrate and either 10 mM MgCl2 or 10 mM MnCl2. The products were resolved by polyacrylamide gel electrophoresis and visualized by autoradiography.

A native gel mobility shift assay was used to examine the binding of PBCV-1 DNA ligase to nicked substrates containing a 32P-labeled DNA or RNA strand on the 5[prime]-phosphate side of the nick. The substrates consisted of a self-complementary 42 nt 3[prime]-OH-terminated hairpin DNA (with a 10 bp stem and an 18 nt 5[prime]-single-strand tail) and a 5[prime]-32P-labeled 18mer strand (either DNA or RNA) annealed to the 5[prime]-tail of the hairpin strand (Fig. 3). Binding reactions were performed in the absence of ATP and divalent cation so as to preclude conversion of substrate to product during the incubation (1). Mixing the ligase with nicked hairpin [32P]DNA resulted in the formation of a discrete protein-DNA complex (Fig. 3). No specific complex was detected when ligase was incubated with the nicked hairpin [32P]RNA ligand. This experiment shows that PBCV-1 DNA ligase discriminates at the substrate binding step between nicked duplexes containing 5[prime]-phosphate DNA versus 5[prime]-phosphate RNA strands.


Figure 3. Native gel assay of the binding of PBCV-1 DNA ligase to nicked substrates with DNA versus RNA 5[prime]-phosphate strands. The nicked hairpin duplex ligand used in the binding assays is shown. The 32P-labeled 18mer strand was either DNA or RNA. Binding reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 250 fmol nicked ligand and 34, 68, 136 or 272 ng PBCV-1 ligase (proceeding left to right within each titration series) were incubated for 10 min at 22°C. Control reactions (lanes -) contained no ligase. The samples were analyzed by native gel electrophoresis. An autoradiogram of the gel is shown.

Ligation of a DNA substrate containing a 5[prime]-ribonucleotide at the nick

Does the failure of PBCV-1 ligase to bind and seal nicked substrates containing a 5[prime]-phosphate RNA strand arise from a stringent requirement for a 5[prime]-deoxynucleotide at the nick? We addressed this question by preparing a nicked hairpin duplex substrate in which the 5[prime]-32P-labeled 18mer contains a single 5[prime]-ribonucleotide in an otherwise all-DNA strand (Fig. 4). Reaction of the 5[prime]-p(rA)-containing substrate with PBCV-1 DNA ligase resulted in joining of the 18mer strand to the 42mer hairpin strand to form a 60mer ligation product (Fig. 4). PBCV-1 DNA ligase sealed the 5[prime]-p(rA) and 5[prime]-p(dA) substrates with comparable efficiencies in the linear range of enzyme dependence (Fig. 5A). The kinetics of ligation were examined under conditions of enzyme excess. The ligation reactions on the 5[prime]-p(rA) and 5[prime]-p(dA) substrates proceeded to 52 and 59% of the end-point values within 5 s (Fig. 5B). We conclude from these experiments that a ribonucleotide at the 5[prime]-phosphate terminus had no effect on strand joining by PBCV-1 ligase.


Figure 4. Ligation at a 5[prime]-ribonucleotide-substituted nicked DNA. The structure of the nicked hairpin substrate used in the ligation reactions is shown. The 32P-labeled 5[prime]-nucleotide at the nick is either 5[prime]-p(dA) or 5[prime]-p(rA). Ligation reaction mixtures (200 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 10 fmol 32P-labeled nicked substrates, 1 mM ATP, 10 mM MgCl2 and 340 ng DNA ligase were incubated at 22°C for 30 min. The reaction products were ethanol precipitated, resuspended in formamide and then separated by electrophoresis through a 12% polyacrylamide gel containing 7 M urea in TBE. The labeled 60mer ligation products were localized by autoradiography and recovered by elution from gel slices soaked in 0.4 ml TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). The eluates were ethanol precipitated. The pellets were resuspended in 50 µl TE. Aliquots containing 1 pmol 32P-labeled 60mer were adjusted to a volume of 13 µl and final concentrations of 0.3 M NaOH, 1 mM EDTA (NaOH +) or 10 mM Tris-HCl, pH 7.5, 1 mM EDTA (NaOH -). These samples were incubated at 37°C for 16 h. Control samples containing the input 18mer DNA substrate that had not been exposed to ligase were treated in parallel (Ligase -). The alkali-treated samples were neutralized by adding 2.2 µl 2 M acetic acid. All samples were then adjusted to 50% formamide, heated for 5 min at 95°C and then analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 M urea in TBE. An autoradiograph of the gel is shown. The positions of 5[prime]-32P-labeled 60mer product and 18mer substrate oligonucleotide are indicated on the left. Alkaline hydrolysis of the DNApRNA ligation product yielded a discrete species, which is denoted by an asterisk.


Figure 5. Rate and extent of ligation at a 5[prime]-ribonucleotide-substituted nicked DNA. (A) Enzyme titration. Ligation reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 500 fmol 5[prime]-32P-(rA) or 5[prime]-32P-(dA) nicked substrate, 1 mM ATP, 10 mM MgCl2 and ligase as specified were incubated for 10 min at 22°C. (B) Kinetics. Ligation reaction mixtures containing (per 20 µl) 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 500 fmol 5[prime]-32P-(rA) or 5[prime]-32P-(dA) nicked substrate, 1 mM ATP, 10 mM MgCl2 and 85 ng ligase were incubated at 22°C. Aliquots (20 µl) were withdrawn at the indicated times and quenched immediately.

In order to prove that PBCV-1 ligase truly catalyzed phospho-diester bond formation to a 5[prime]-ribonucleotide, we analyzed the susceptibility of the gel-purified 60mer DNAp(rA)DNA ligation product to treatment with NaOH. The gel-purified 60mer product of 3[prime]-OH DNA to [5[prime]-32P]DNA ligation was analyzed in parallel, along with the input 18mer strands. The [5[prime]-32P](dA) 18mer strand was unaffected by alkali, whereas the [5[prime]-32P](rA) 18mer was hydrolyzed completely (Fig. 4). (The predicted 32P-labeled hydrolysis products, adenosine 5[prime],3[prime]-bisphosphate and adenosine 5[prime],2[prime]-bisphosphate, run off the gel and are not seen in Fig. 4.) The internally labeled 60mer DNAp(dA)DNA ligation product was unaffected by NaOH (Fig. 4). However, the 60mer DNAp(rA)DNA ligation product was converted quantitatively to the expected product, 5[prime]-TGTAGTCACTATCGGAATAAGGGCGACATTTTTGTCGCCCTTp(rA)p (denoted by the asterisk in Fig. 4).

Effects of base mismatches at the reactive 5[prime]-phosphate and 3[prime]-hydroxyl positions

The experiments presented above indicated that PBCV-1 ligase was indifferent to ribose sugar substitutions at the 3[prime]-OH and the 5[prime]-phosphate nucleotides at the nick. To gain additional insights into the enzyme's substrate specificity and fidelity, we examined the effects of single base mismatches at the nick. Sets of four otherwise identical 18mer oligonucleotides containing dC, dG, dA and dT at their 5[prime]-ends were 5[prime]-32P-labeled and annealed with various 3[prime]-OH-terminated 18mer and 36mer complementary strands to form nicked duplexes with all 12 possible 5[prime] mismatches (Fig. 6). Mismatch effects on ligation were gauged by the extent of formation of the 36mer ligation products as a function of enzyme concentration. The specific activities of PBCV-1 ligase in sealing the various 5[prime]-phosphate mispaired nicks are tabulated in Figure 6 and are expressed as a percentage of the specific activity in ligating the base paired control substrate in each series of four nicked duplexes. PBCV-1 ligase was fairly tolerant of most of the 5[prime] mismatches tested, with the exception of the 5[prime]-A:G and 5[prime]-G:A mispaired DNAs, for which the specific activity of strand joining was <1% of the paired 5[prime]-C:G and 5[prime]-T:A controls (Fig. 6B). Activity at a 5[prime]-A:A mismatch was 10% of the paired control.


Figure 6. Effects of 5[prime]-phosphate base mismatches on strand joining. Nicked duplex DNA substrates were prepared by annealing a 5[prime]-32P-labeled 18mer strand and an unlabeled 3[prime]-OH 18mer oligonucleotide to a 36mer complementary strand. The structures of duplexes with mismatches at the 5[prime]-phosphate side of the nick are shown in (A)-(D). Mismatch nucleotides are indicated in lower case. Ligation reaction mixtures containing 500 fmol radiolabeled substrate and increasing amounts of PBCV-1 ligase (typically 0.4, 0.8, 1.7, 3.4 and 6.8 ng) were incubated for 10 min at 22°C. The specific activity was determined from the slope of the titration curve in the linear range of enzyme dependence. (Each specific activity value was the average of two titration experiments.) The ligase activity on each of the indicated mispaired substrates was then normalized to the specific activity on the fully base paired control substrate (defined as 100%).

A separate series of nicked duplex ligation substrates contained a single mispaired base at the reactive 3[prime]-OH of the nick (Fig. 7). Significant inhibition (which we define as at least an order of magnitude decrement in specific activity) was observed with 3[prime]-G:A, 3[prime]-G:T, 3[prime]-G:G, 3[prime]-T:T, 3[prime]-A:G, 3[prime]-A:C and 3[prime]-C:C mispairs. The results indicate that 3[prime]-G:N mispairs are uniformly poor substrates for PBCV-1 ligase.


Figure 7. Effects of 3[prime]-OH base mismatches on strand joining. Substrates were prepared by annealing a 5[prime]-32P-labeled 18mer strand and an unlabeled 3[prime]-OH 18mer oligonucleotide to a 36mer complementary strand. The structures of duplexes with mismatches on the 3[prime]-OH side of the nick are shown in (A)-(D). Mismatch nucleotides are indicated in lower case. Ligase specific activity was determined as described in the legend to Figure 6.

DISCUSSION

Discrimination of RNA versus DNA strands

Our experiments show that PBCV-1 DNA ligase is capable of joining a 3[prime]-OH RNA strand to a 5[prime]-phosphate DNA, but is unable to ligate DNA to RNA or RNA to RNA when the polynucleotides comprising the nick are annealed to a DNA template. PBCV-1 ligase also cannot ligate DNA molecules annealed to an RNA template.

The instructive point is that PBCV-1 DNA ligase is unreactive with substrates that contain an RNA strand on the 5[prime]-phosphate side of the nick, regardless of whether the chemically reactive `top strand' of the double helix (the 5[prime]-phosphate strand) or the `bottom' template strand is composed of RNA. The suppressive effects of RNA-DNA hybrid formation on ligation suggest that the nicked substrate must adopt a B-form helical conformation on the 5[prime]-phosphate side in order to be sealed. The RNA strand of an RNA-DNA hybrid adopts the A-form helical conformation (as found in double-strand RNA), while the DNA strand adopts a conformation that is intermediate in character between A and B forms (13,14). PBCV-1 DNA ligase is nick-specific, but not sequence-specific. Hence, we assume that the protein makes non-specific contacts with the phosphodiester backbone of the duplex in addition to specific contacts with the reactive 3[prime]-OH and 5[prime]-phosphate ends. Adoption of non-B conformation may weaken or preclude these backbone contacts. Indeed, a native gel shift experiment confirms that PBCV-1 DNA ligase binds stably to the nicked ligand containing a 5[prime]-phosphate DNA strand, but not to a nicked duplex containing a 5[prime]-phosphate RNA strand. Because PBCV-1 DNA ligase does join 3[prime]-OH RNA to 5[prime]-phosphate DNA, we infer that a B-form helical conformation on the upstream side of the nick is not essential for substrate recognition or reaction chemistry.

Vaccinia DNA ligase, mouse L cell DNA ligase (presumably DNA ligase I) and Escherichia coli DNA ligase display the same selectivity as the PBCV-1 enzyme in catalyzing the joining of 3[prime]-OH RNA to 5[prime]-phosphate DNA, but not 3[prime]-OH DNA to 5[prime]-phosphate RNA (12,23,24). The overall specificity of PBCV-1 ligase in RNA strand joining is most similar to that of vaccinia virus DNA ligase (see 12 for a review of the ability of ATP-dependent DNA ligases to seal RNA-containing substrates). A subtle distinction between the two eukaryotic viral ligases is that the vaccinia enzyme can ligate 3[prime]-OH DNA to 5[prime]-phosphate RNA at low efficiency in the presence of manganese (12).

Embedding of single ribonucleotides in DNA

Ribonucleotide substitution at the 5[prime]-phosphate of an otherwise all-DNA nicked duplex has no significant effect on the rate or extent of phosphodiester bond formation by PBCV-1 DNA ligase. Thus, the inability of the PBCV-1 enzyme to join a 3[prime]-OH DNA strand to a 5[prime]-phosphate RNA strand cannot be attributed to the mere presence of a ribonucleotide at the nick. Vaccinia virus DNA ligase can also efficiently seal the 5[prime]-p(rA)-substituted nicked duplex substrate (our unpublished data). Rumbaugh et al. (15) recently reported that purified recombinant human DNA ligase I can join a 3[prime]-OH DNA strand to a singly ribo-substituted 5[prime]-p(rA)-DNA oligonucleotide annealed to a template DNA strand. However, the specific activity of ligase I in sealing the 5[prime]-p(rA)-substituted nicked duplex was 3% of its specific activity on an all-DNA nicked substrate of identical sequence (15). Thus, the Chlorella virus and vaccinia virus enzymes appear to be considerably more tolerant of ribo substitution at the reactive 5[prime]-phosphate than human DNA ligase I. The data of Rumbaugh et al. (15) show that reaction of ligase I with 5[prime]-p(rA)-substituted nicked DNA results in accumulation of very high levels of the normally evanescent adenylated nucleic acid intermediate. We observed no accumulation of the adenylated intermediate during the sealing of a 3[prime]-OH/5[prime]-p(rA) nicked substrate by either Chlorella virus or vaccinia virus DNA ligase.

The result of 3[prime]-OH/5[prime]-p(rA) nick joining by DNA ligase is the embedding of a monoribonucleotide in DNA. Rumbaugh et al. (15) propose that ribonucleotide embedding can occur during the processing of RNA-primed Okazaki fragments. They also suggest that embedded ribonucleotides can be removed from eukaryotic cellular DNA by the concerted action of nucleases RNase H1 and FEN-1 (15). Assuming that the facility of ribonucleotide embedding by Chlorella virus and vaccinia virus DNA ligases in vitro applies in vivo [i.e. that 3[prime]-OH/5[prime]-p(rN) nicked substrates are available in virus-infected cells], one might expect these DNA viruses to encode surveillance enzymes that recognize and remove embedded ribonucleotides from the viral genome. Chlorella virus does encode a putative repair endonuclease that resembles bacteriophage T4 endonuclease V, a second putative endonuclease related to Bacillus subtilis phage endonucleases and a putative exonuclease resembling [lambda] exonuclease (16-18). Sekiguchi and Shuman (19) have suggested that vaccinia virus and eukaryotic cells may employ type IB topoisomerases to survey duplex DNA for embedded ribonucleotides, i.e. by topoisomerase-mediated endonucleolytic cleavage at sites of ribo substitution to leave a 2[prime],3[prime]-cyclic phosphate/5[prime]-OH nick that cannot be rejoined by conventional polynucleotide ligases. Although the physiological burden of uncorrected ribonucleotide incorporation into cellular and viral DNA is not well understood, the available data implicate DNA ligases in single ribonucleotide embedding and suggest that there are multiple pathways by which embedded ribonucleotides might be recognized and marked for removal.

Ligase fidelity

Fidelity of strand joining refers to the extent to which the enzyme can ligate substrates containing mismatched bases on either side of the nick. Here, we tested the effects of all 12 5[prime]-phosphate mismatches and all 12 3[prime]-OH mismatches on ligase-specific activity. PBCV-1 ligase was fairly tolerant of 5[prime]-phosphate mismatches, except for the 5[prime]-A:G and 5[prime]-G:A mispairs, which elicited a 10-2 decrement in activity compared with the correctly paired 5[prime]-C:G and 5[prime]-T:A controls. The capacity to discriminate against the 5[prime]-A:G mispair distinguishes the Chlorella virus enzyme from vaccinia virus ligase; the specific activity of vaccinia ligase on the 5[prime]-A:G nick was 70% of its activity on the paired 5[prime]-C:G control (our unpublished data). Tomkinson et al. (20) found that Saccharomyces cerevisiae DNA ligase I effectively sealed five different 5[prime] mispair configurations: 5[prime]-T:C, 5[prime]-A:C, 5[prime]-T:A, 5[prime]-G:A and 5[prime]-C:A. The activity of yeast ligase I on a 5[prime]-A:G substrate was not examined in their study. Husain et al. (21) reported that mammalian DNA ligases I and III were active in sealing the same 5[prime] mispaired substrates used in the analysis of the yeast enzyme. We surmise that PBCV-1 ligase has higher fidelity than ligases I and III with respect to a 5[prime]-G:A mispair. It is not yet clear if eukaryotic cellular ligases I and III are vaccinia-like or PBCV-1-like in their reactivity with a 5[prime]-A:G mispaired nick. Luo and Barany (22) tested the fidelity of the NAD-dependent Thermus thermophilus DNA ligase in joining all 12 possible 5[prime]-phosphate mispaired nicked duplexes. They found that Tth ligase was incapable of sealing 5[prime]-A:G, 5[prime]-C:C, 5[prime]-T:C and 5[prime]-G:G mispairs (22).

The Chlorella virus ligase resembles other DNA ligases insofar as it is generally more sensitive to 3[prime]-OH mispairs than to 5[prime]-phosphate mispairs (except for A:G and G:A). Yet, the hierarchy of 3[prime]-OH mismatch effects appears to differ significantly from one ligase to the next. For example, a 3[prime]-A:A mispair is sealed by PBCV-1 ligase, but is virtually inert as a substrate for vaccinia ligase and Tth ligase (11,22). PBCV-1 ligase, like mammalian ligase I, is inhibited by a 3[prime]-G:T mispair (21), yet the homologous ligase I of yeast readily seals a 3[prime]-G:T mismatch (20). There are some consistent findings, however. For example, Chlorella virus ligase, vaccinia ligase and Tth ligase are inhibited by 3[prime]-G:G and 3[prime]-T:T mispairs.

The theme underscored by this and other studies of eukaryotic, viral and cellular ligases is that each enzyme displays a distinct pattern of fidelity in strand joining. The fact that Chlorella virus PBCV-1 DNA ligase is reasonably active on 14 of 24 mismatch configurations tested suggests that this enzyme may contribute to the generation of certain missense mutations by ligating strands containing mispairs on either side of a nick. This pathway of mutagenesis has been discussed previously for yeast and vaccinia DNA ligases (11,20). The structural basis for mismatchdiscrimination by DNA ligase is obscure. The crystal structure of T7 DNA ligase (8) has illuminated determinants of ATP binding which are conserved in the ATP-dependent ligases of bacteriophages, archaea, certain eubacteria, eukaryotic cells and eukaryotic DNA viruses. However, we know virtually nothing about how DNA ligases recognize and bind to a nicked DNA duplex. The challenge now is to determine the crystal structure of DNA ligase bound at a nick.

REFERENCES

1. Ho,C.K., Van Etten,J.L. and Shuman,S. (1997) J. Virol., 71, 1931-1937. MEDLINE Abstract

2. Lehman,I.R. (1974) Science, 186, 790-797. MEDLINE Abstract

3. Engler,M.J. and Richardson,C.C. (1982) Enzymes, 15, 3-29.

4. Tomkinson,A.E. and Levin,D.S. (1997) BioEssays, 19, 893-901. MEDLINE Abstract

5. Tomkinson,A.E., Totty,N.F., Ginsburg,M. and Lindahl,T. (1991) Proc. Natl. Acad. Sci. USA, 88, 400-404. MEDLINE Abstract

6. Kodama.K., Barnes,D.E. and Lindahl,T. (1991) Nucleic Acids Res., 19, 6093-6099.

7. Shuman,S. and Ru,X. (1995) Virology, 211, 73-83. MEDLINE Abstract

8. Subramanya,H.S., Doherty,A.J., Ashford,S.R. and Wigley,D.B. (1996) Cell, 85, 607-615. MEDLINE Abstract

9. Sriskanda,V. and Shuman,S. (1998) Nucleic Acids Res., 26, 525-531. MEDLINE Abstract

10. Shuman,S. and Schwer,B. (1995) Mol. Microbiol., 17, 405-410. MEDLINE Abstract

11. Shuman,S. (1995) Biochemistry, 34, 16138-16147. MEDLINE Abstract

12. Sekiguchi,J. and Shuman,S. (1997) Biochemistry, 36, 9073-9079. MEDLINE Abstract

13. Arnott,S., Chandrasekaran,R., Millane,R.P. and Park,H. (1986)J. Mol. Biol., 188, 631-640. MEDLINE Abstract

14. Salazar,M., Federoff,O.Y., Miller,J.M., Ribeiro,N.S. and Reid,B.R. (1993) Biochemistry, 32, 4207-4215. MEDLINE Abstract

15. Rumbaugh,J.A., Murante,R.S., Shi,S. and Bambara,R.A. (1997)J. Biol. Chem., 272, 22592-22599.

16. Lu,Z., Li,Y., Zhang,Y., Kutish,G.F., Rock,D.L. and Van Etten,J.L. (1995) Virology, 206, 339-352. MEDLINE Abstract

17. Kutish,G.F., Li,Y., Lu,A., Furuta,M., Rock,D.L. and Van Etten,J.L. (1996) Virology, 223, 303-317. MEDLINE Abstract

18. Li,Y., Lu,Z., Burbank,D.E., Kutish,G.F., Rock,D.L. and Van Etten,J.L. (1995) Virology, 212, 134-150. MEDLINE Abstract

19. Sekiguchi,J. and Shuman,S. (1997) Mol. Cell. Biol., 1, 89-97.

20. Tomkinson,A.E., Tappe,N.J. and Friedberg,E.C. (1992) Biochemistry, 31, 11762-11771. MEDLINE Abstract

21. Husain,I., Tomkinson,A.E., Burkhart,W.A., Moyer,M.B., Ramos,W., Mackey,Z.B., Besterman,J.M. and Chen,J. (1995) J. Biol. Chem., 270, 9683-9690. MEDLINE Abstract

22. Luo,J. and Barany,F. (1996) Nucleic Acids Res., 24, 3079-3085. MEDLINE Abstract

23. Bedows,E., Wachsman,J.T. and Gumport,R.I. (1977) Biochemistry, 16, 2231-2235. MEDLINE Abstract

24. Nath,K. and Hurwitz,J. (1974) J. Biol. Chem., 249, 3680-3688. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +1 212 639 7145; Fax: +1 212 717 3623; Email: s-shuman{at}ski.mskcc.org

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