©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of DNA Ligase III from Bovine Testes
HOMOLOGY WITH DNA LIGASE II AND VACCINIA DNA LIGASE (*)

Intisar Husain (1)(§), Alan E. Tomkinson (3), William A. Burkhart (2), Mary B. Moyer (2), William Ramos (3), Zachary B. Mackey (3), Jeffrey M. Besterman (1), Jingwen Chen (1)(§)(¶)

From the (1) Department of Cell Biology, (2) Department of Bioanalytical and Structural Chemistry, Glaxo Research Institute, Research Triangle Park, North Carolina 27709 and the (3) Institute of Biotechnology, Center for Molecular Medicine, The University of Texas Health Science Center, San Antonio, Texas 78245

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mammalian cell nuclei contain three biochemically distinct DNA ligases. In the present study we have found high levels of DNA ligase I and DNA ligase III activity in bovine testes and have purified DNA ligase III to near homogeneity. The high level of DNA ligase III suggests a role for this enzyme in meiotic recombination. In assays measuring the fidelity of DNA joining, we detected no significant differences between DNA ligases II and III, whereas DNA ligase I was clearly a more faithful enzyme and was particularly sensitive to 3` mismatches. Amino acid sequences of peptides derived from DNA ligase III demonstrated that this enzyme, like DNA ligase II, is highly homologous with vaccinia DNA ligase. The absence of unambiguous differences between homologous peptides from DNA ligases II and III (10 pairs of peptides, 136 identical amino acids) indicates that these enzymes are either derived from a common precursor polypeptide or are encoded from the same gene by alternative splicing. Based on similarities in amino acid sequence and biochemical properties, we suggest that DNA ligases II and III, Drosophila DNA ligase II, and the DNA ligases encoded by the pox viruses constitute a distinct family of DNA ligases that perform specific roles in DNA repair and genetic recombination.


INTRODUCTION

DNA joining is required to link together Okazaki fragments during lagging strand DNA synthesis and to seal DNA strand breaks produced either by the direct action of a damaging agent or by DNA repair enzymes removing DNA lesions. In addition, DNA ligation is necessary to complete exchange events between homologous duplex DNA molecules. Prokaryotes contain a single species of DNA ligase that presumably functions in each of the above DNA metabolic pathways (1) . In contrast, three biochemically distinct DNA ligases have been identified in extracts from mammalian cells (2) .

In in vitro assays DNA ligase I appears to be the enzyme that joins Okazaki fragments during DNA replication (3, 4, 5) . The abnormal pattern of DNA replication intermediates detected in experiments with the human cell line 46BR and its derivatives, which contain mutated DNA ligase I alleles, are consistent with an in vivo defect in Okazaki fragment joining (6, 7, 8, 9) . Furthermore, the sensitivity of these cell lines to DNA damaging agents suggests that DNA ligase I may also be involved in certain DNA repair pathways (6, 10, 11, 12) .

The high levels of DNA ligase I activity in the thymus of young animals facilitated the purification of this enzyme to homogeneity from calf thymus glands (13, 14) . Two minor DNA ligase activities, designated as DNA ligase II and DNA ligase III, have also been identified in calf thymus extracts (2, 15, 16) . The 70-kDa DNA ligase II, which is the major DNA joining activity in the normal liver (17) , is not recognized by a polyclonal antiserum specific for DNA ligase I (2, 15, 18, 19) . Recent amino acid sequencing studies with homogeneous bovine DNA ligase II confirmed that this enzyme is not a proteolytic fragment of DNA ligase I and revealed that the enzyme is highly homologous with the DNA ligase encoded by vaccinia virus (18) . It has been reported that the level of DNA ligase II activity is induced by DNA damage, suggesting that it may play a role in DNA repair (20, 21) .

The 100-kDa DNA ligase III is also not recognized by the DNA ligase I-specific antiserum (2) . However, the relationship between DNA ligase II and DNA ligase III is less clearly defined. Based on differences in the physical, catalytic, and chromatographic properties of these enzymes, it was concluded that they are probably encoded by separate genes (2) . In contrast, a recent comparison of DNA ligase adenylation sites by peptide mapping demonstrated significant similarities between the active sites of these enzymes, suggesting that they may be related by alternative splicing (22) . The association of DNA ligase III with a calf thymus recombination complex (23) and with a human DNA repair protein, XRCC1 (24) , is consistent with this enzyme joining DNA strand breaks to complete recombination and repair events.

In this report we describe the purification of DNA ligase III to near homogeneity from bovine testes. Amino acid sequencing studies have revealed a high degree of homology between DNA ligase III and vaccinia DNA ligase. Furthermore, many of the DNA ligase III peptides were identical with peptides isolated from bovine DNA ligase II. The absence of unambiguous differences between homologous DNA ligase II and III peptides indicates that these enzymes are either derived from a common precursor polypeptide or are encoded from the same gene by alternative splicing.


MATERIALS AND METHODS

Purification of Recombinant Human DNA Ligase I

Human DNA ligase I cDNA was subcloned into a baculovirus expression vector, pVL1392 (PharMingen). The details of the purification of recombinant human DNA ligase I from baculoviral-infected insect cells will be described elsewhere. In assays with the oligo(dT)/poly(dA) substrate, homogeneous 125-kDa DNA ligase I had a specific activity of 2.5 units/mg.

Partial Purification of DNA Ligase I and DNA Ligase III from Whole Cell Extracts of Bovine Testes

Testes from newly slaughtered bulls were kept on ice and processed within 3 h. A cell-free extract was prepared from 250 g of bovine testes by homogenization and then fractionated by phosphocellulose chromatography, ammonium sulfate precipitation, and gel filtration as described by Tomkinson et al. (2) . Protein concentrations were measured by the method of Bradford (25) . Fractions eluting from the gel filtration column were assayed for DNA joining activity with both the oligo(dT)/poly(dA) and oligo(dT)/poly(rA) substrates and for enzyme-adenylate formation. Fractions containing both DNA ligase I and DNA ligase III activity (2, 8) were pooled and fractionated by hydroxylapatite chromatography (26) . Consistent with previous results, the majority of DNA ligase I activity was eluted by 150 m M potassium phosphate. DNA ligase I was further purified by native DNA cellulose chromatography and FPLC() Mono Q chromatography as described (14) and was approximately 30% homogeneous.

DNA ligase III activity, which was eluted by 400 m M potassium phosphate, was dialyzed against 50 m M Tris-HCl (pH 7.5), 50 m M NaCl, 1 m M EDTA, 0.5 m M DTT, 10% glycerol (buffer A) and applied to a native DNA cellulose column. Bound proteins were eluted stepwise with 0.2 and 0.5 M NaCl in buffer A. Active fractions, which eluted with 0.5 M NaCl, were dialyzed against buffer A and then applied to an FPLC Mono Q 5/5 column. Bound proteins were eluted with a 20-ml linear gradient from 0.05-0.75 M NaCl in buffer A. DNA ligase III activity eluted at 250 m M NaCl. A 100-kDa polypeptide detected by Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis co-eluted with DNA ligase activity. Assuming that this polypeptide was responsible for the labeled 100-kDa enzyme-adenylate, this preparation of DNA ligase III was approximately 30% homogeneous.

For amino acid sequencing studies, the peak fractions of DNA ligase III from the FPLC Mono Q column (2 ml, 25 µg) were pooled and concentrated by ultrafiltration using a Centricon-10 apparatus (Amicon) that had been pretreated with 2% Triton X-100. Polypeptides (400 µl) were separated by electrophoresis through a preparative 10% SDS-polyacrylamide gel and then transferred to a polyvinylidene membrane (Bio-Rad). After staining with Ponceau S, the strip of membrane containing the 100-kDa DNA ligase III was excised and washed with distilled H0. After digestion in situ with trypsin, the resultant peptides were separated by reverse phase HPLC (27) .

Purification of DNA Ligase III from Testis Nuclei

Three testes (0.75 kg) were sliced into 1-inch cubes, resuspended in 1 liter of buffer B (50 m M Tris-HCl (pH 7.5), 0.25 M sucrose, 2 m M MgCl, 10 m M -mercaptoethanol, 0.8 m M phenylmethylsulfonyl fluoride, 0.2 m M Pefabloc (Boehringer Mannheim), 2 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 3.5 µg/ml TPCK, 25 µg/ml TLCK, and 1 m M benzamidine) and homogenized in a Waring blender. The homogenate was filtered through cheesecloth with buffer B added periodically to maintain a volume of about 1.5 liters. Nuclei were collected by centrifugation at 2500 g for 30 min and washed three times with buffer B.

The crude nuclei (40 g) were resuspended in 50 m M Tris-HCl (pH 7.5), 1 m M EDTA, 750 m M NaCl, 10% glycerol, 10 m M -mercaptoethanol, 1 m M Pefabloc, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 5 µg/ml chymostatin, 3.5 µg/ml TPCK, 25 µg/ml TLCK, and 1 m M benzamidine (buffer C) and then lysed by Dounce homogenization. After the addition of 40% polyethylene glycol 8000 to a final concentration of 5%, the suspension was stirred for 15 min and then centrifuged at 10,000 rpm for 10 min in a GSA rotor (Sorvall). The clarified nuclear extract (160 ml, 247 mg) was adjusted to 1 m M potassium phosphate and then loaded onto a 35-ml hydroxylapatite column that had been equilibrated with buffer C containing 1 m M potassium phosphate. Proteins were eluted stepwise with 50, 150, and 400 m M potassium phosphate (pH 7.5) buffers containing 1.0 m M DTT and protease inhibitors as described in buffer C. DNA ligase III activity, which was eluted in the 400 m M fraction (30 ml, 90 mg), was diluted 1 in 4 with 67 m M NaCl, 1.33 m M EGTA, and 1.33 m M DTT and loaded onto a 6.5-ml P11 phosphocellulose column that had been equilibrated with buffer D (50 m M Tris-HCl (pH 7.5), 50 m M NaCl, 1 m M EDTA, 1 m M EGTA, 10% glycerol, 1 m M DTT, and protease inhibitors as described in buffer C). Bound proteins were eluted stepwise with 100, 250, and 450 m M NaCl sequentially in buffer D. DNA ligase III activity was detected in the 450 m M eluate (12 ml, 22 mg). The samples were then diluted 1 in 6 with buffer D without NaCl to adjust the NaCl to 75 m M and loaded onto a 5-ml native DNA-cellulose column equilibrated with buffer D. Bound proteins were eluted stepwise with buffer D containing 200 m M and 500 m M NaCl. DNA ligase III activity (9 ml, 5 mg), which was eluted in the 500 m M NaCl buffer, was loaded onto an AcA34 gel filtration column (2.6 98 cm) that had been equilibrated with buffer D containing 1 M NaCl. Active fractions were pooled, dialyzed against buffer D, and then loaded onto an FPLC Mono Q HR 5/5 column. Bound proteins were eluted with a 20 ml of linear gradient from 50 to 750 m M NaCl in buffer D. DNA ligase III (0.7 ml, 35 µg), which eluted at about 250 m M NaCl, was stored in aliquots at -80 °C. Under these storage conditions, the enzyme was stable for at least 6 months.

Preparation of Substrates for DNA Joining Assays

Polynucleotides dA, rA, and dT were purchased from Pharmacia Biotech Inc. Oligo(dT)was synthesized on an Applied Biosystems model 392 DNA/RNA synthesizer. Labeled homopolymer substrates were prepared as described previously (28) . Labeled polynucleotide substrates containing a single, defined nick were prepared by annealing three complementary oligonucleotides as described previously (28) .

DNA Ligase Assays

Phosphodiester bond formation was assayed as described previously (28) . One unit of DNA ligase activity catalyzes the conversion of 1 nmol of terminal phosphate residues to a phosphatase-resistant form in 15 min at 20 °C.

Analysis of Ligation Products

Aliquots (10 µl) from DNA ligase assays were added to 10 µl of formamide dye and heated for 2 min at 90 °C. Samples (2.5 µl) were then loaded onto a denaturing 10% polyacrylamide gel. After electrophoresis, the gels were dried and oligonucleotides were visualized by autoradiography. Formation of ligated products was quantitated by phosphorimage analysis (Molecular Dynamics).

Formation of DNA Ligase-Adenylate

The adenylation reactions (12 µl) were routinely carried out in a reaction mixture containing 60 m M Tris-HCl (pH 7.5), 10 m M MgCl, 5 m M DTT, 50 µg/ml bovine serum albumin, 0.5-3.0 µCi [-P] ATP (3000 Ci/mmol, Amersham Corp.) and the enzyme fraction (29) . After incubation at room temperature for 15 min, reactions were stopped by the addition of an equal volume of 2 SDS sample buffer. Samples were heated at 90 °C for 5 min and polypeptides were separated by electrophoresis through an 8% SDS-polyacrylamide gel (30) . Gels were fixed in 10% acetic acid and dried. Adenylylated polypeptides were detected by autoradiography.

Immunoblotting

Proteins were separated by denaturing polyacrylamide gel electrophoresis (30) and transferred to nitrocellulose membranes. After incubation with either antiserum raised against homogeneous bovine DNA ligase I (14) or antiserum raised against the conserved COOH-terminal peptide of eukaryotic DNA ligases (14) , antigen-antibody complexes were detected by enhanced chemiluminescence (Amersham).

Proteolytic Digestion and Amino Acid Sequencing of Bovine DNA Ligase III Peptides

Peptide sequences were obtained from both the partially purified and the near homogeneous preparations of DNA ligase III. DNA ligase III peptides from the partially purified preparation were isolated as described above. Near homogeneous DNA ligase III (10-15 µg) was applied to a hydrophobic sequencing column (Hewlett-Packard) according to the manufacturer's instructions. After in situ digestion with endoproteinase Lys-C (Wako), peptides were separated by reverse phase HPLC using a Spheri 5 ODS (Brownlee) column (27) . The amino acid sequences of peptides were determined by automated Edman degradations performed on the ABI477A protein sequencer with the 120A phenylthiohydantoin analyzer.


RESULTS

Partial Purification of DNA Ligase I and DNA Ligase III from Whole Cell Extracts of Bovine Testes

Three biochemically distinct DNA ligase activities have been identified in whole cell extracts from calf thymus glands (2) . Since the high levels of DNA ligase I activity in this tissue hinders the purification of DNA ligases II and III, we have examined the relative levels of the DNA ligases in other bovine tissues. Recently, we have described the purification of DNA ligase II to homogeneity from liver nuclei (18) . We did not detect DNA ligase III in significant quantities in liver extracts, and, therefore, we investigated the levels of DNA ligase III in testes. In order to compare the relative levels of DNA ligase III in the thymus and testes, we employed the same fractionation procedure used to purify DNA ligase III from calf thymus glands (2) . After separation by gel filtration, a major peak of high molecular weight DNA joining activity containing both DNA ligase I and DNA ligase III was detected in assays with both the oligo(dT)/poly(dA) and oligo(dT)/poly(rA) substrates. Since DNA ligase I is not active with the oligo(dT)/poly(rA) substrate (2) , the joining activity measured with this substrate reflects DNA ligase III activity. The specific activity of DNA ligase III was 4-5-fold higher in fractions from the testes compared with similar fractions from calf thymus glands (2) , demonstrating that the testes contain significantly higher levels of DNA ligase III.

To determine the relative contribution of DNA ligase I and DNA ligase III to the high molecular weight DNA joining activity, the pooled fractions from the gel filtration column were fractionated by hydroxylapatite chromatography. Consistent with previous observations (14, 26) , the majority of DNA ligase I was eluted with 150 m M potassium phosphate, whereas DNA ligase III was eluted with 400 m M potassium phosphate. The 400 m M eluate contained approximately 2-fold more DNA joining activity, measured with the oligo(dT)/poly(dA) substrate, than the 150 m M eluate. Thus, it appears that DNA ligase III is a major DNA joining activity in the testes.

Purification of DNA Ligase III from Testis Nuclei

Although DNA ligase I is a nuclear enzyme (31) , this enzyme rapidly leaks out of nuclei during subcellular fractionation and is mainly found in the cytoplasmic/soluble fraction (19) . In contrast, DNA ligase II remains firmly associated with nuclei isolated under isotonic conditions (18, 19, 32) . The majority of DNA ligase III activity also remains associated with similarly prepared nuclei from bovine testes. In assays measuring enzyme-adenylate formation, the major labeled product in testis nuclear extracts corresponds to the 100-kDa DNA ligase III, with the 125-kDa DNA ligase I contributing about 5% and the 70-kDa DNA ligase II less than 1% (data not shown). Similarly prepared nuclear extracts from bovine liver also contain low levels of DNA ligase I, but in this tissue, the 70-kDa DNA ligase II is the predominant enzyme (18) .

DNA ligase III was purified to greater than 90% homogeneity from testis nuclear extracts by monitoring formation of the 100 kDa enzyme-adenylate intermediate and joining of the oligo(dT)/poly(dA) substrate. After the final FPLC Mono Q column, a single major band with an apparent molecular mass of 100 kDa co-eluted with DNA joining activity. Analysis of the protein content of the peak fraction by Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis detected a minor polypeptide with an apparent molecular mass of 87 kDa in addition to the major band at 100 kDa (Fig. 1, lane 1). In assays measuring enzyme-adenylate formation, labeled products of 100 and 87 kDa were generated in the same relative amounts as the polypeptides stained with Coomassie Blue (Fig. 1, lane 2). This 87-kDa polypeptide is probably the active proteolytic fragment of DNA ligase III described previously (2) . Approximately 35 µg of the 100 kDa form of DNA ligase III were obtained from 750 g of bovine testes.


Figure 1: Analysis of purified bovine DNA ligase III by SDS-polyacrylamide gel electrophoresis. Polypeptides were separated by electrophoresis through an 8% SDS-polyacrylamide gel. Lane 1, the peak fraction of DNA ligase III (400 ng) from testis nuclei after FPLC Mono S chromatography. Proteins were detected by staining with Coomassie Brilliant Blue; lane 2, 50 ng of the same fraction was assayed for enzyme-adenylate formation as described under ``Materials and Methods.'' The positions of size markers, 97-kDa phosphorylase b, 66-kDa bovine serum albumin, and 45-kDa ovalbumin (Bio-Rad) are indicated on the left.



In DNA joining assays, the most highly purified fractions had a specific activity of 2 units/mg with the oligo(dT)/poly(dA) substrate and 0.2 unit/mg with the oligo(dT)/poly(rA) substrate. The value measured with the DNA/DNA substrate is similar to that obtained for homogeneous bovine DNA ligase I (2.5 units/mg) (14) , recombinant human DNA ligase I (2.5 units/mg), and bovine DNA ligase II (2 units/mg) (18) .

Bovine DNA Ligase III Is Recognized by the Antiserum Raised against the COOH-terminal Peptide Sequence Conserved in Eukaryotic DNA Ligases

To confirm that the putative DNA ligase III polypeptides were not derived from 125 kDa DNA ligase I by proteolysis, we performed immunoblotting experiments with the antiserum raised against homogeneous bovine DNA ligase I. As reported previously (2) , this antiserum does not cross-react with partially purified DNA ligase III (Fig. 2 A). However, both the 100- and 87-kDa DNA ligase III polypeptides are recognized by the antiserum raised against a conserved COOH-terminal peptide sequence (Fig. 2, B and C) that was originally identified in a comparison of Saccharomyces cerevisiae Cdc9 DNA ligase, Schizosaccharomyces pombe Cdc17 DNA ligase and vaccinia DNA ligase (33) . Subsequently, homologous peptide sequences have been found in mammalian DNA ligase I (14, 34) and DNA ligase II (18) . The conservation of this peptide sequence in all eukaryotic DNA ligases presumably indicates that it plays an important but as yet undefined role in the catalytic function of these enzymes.


Figure 2: Bovine DNA ligase III cross-reacts with the antiserum raised against the conserved epitope present in all eukaryotic DNA ligases but not with an antiserum raised against bovine DNA ligase I. Polypeptides were separated by electrophoresis through an 8% SDS-polyacrylamide gel and then transferred to nitrocellulose membranes. A, lane 1, partially purified DNA ligase I from testis whole cell extracts, 100 ng of 125-kDa polypeptide; lane 2, homogeneous DNA ligase II from bovine liver, 100 ng (18); lane 3, partially purified DNA ligase III from testis whole cell extracts, 50 ng of 100-kDa polypeptide. The membrane was incubated with antiserum raised against homogeneous DNA ligase I (14). B, proteins in lanes 4-6 are identical to those in lanes 1-3 except that the membrane was incubated with antiserum raised against a peptide common to all eukaryotic DNA ligases (14). C, lane 7, the peak fraction of DNA ligase III from testis nuclei after FPLC Mono S chromatography (400 ng). The membrane was incubated with the same antiserum as in B. Immune complexes were detected by enhanced chemiluminescence. The positions of the three DNA ligases are indicated. The 87-kDa band is probably an active proteolytic fragment of DNA ligase III (2).



Reactivity of DNA Ligases I, II, and III with Polynucleotide Substrates Containing a Single Defined Nick

The three mammalian DNA ligases can be distinguished by their reactivity with different homopolymer substrates (2) , but these differences in substrate specificity may not be physiologically significant. Consequently, we have examined the reactivity of the three mammalian DNA ligases with DNA molecules containing a single, defined nick that more closely resembles the in vivo substrate. Consistent with previous studies on S. cerevisiae Cdc9 DNA ligase (28) , the efficiency of DNA joining by the mammalian DNA ligases was not significantly affected by 5`-mismatched termini (data not shown). Using DNA substrates with 3`-mismatched termini opposite a pyrimidine, DNA ligase III was not significantly inhibited by a 3`C/T mismatch, but a 3`G/T mismatch reduced the amount of ligated product by about 5-fold (Fig. 3 B). DNA ligase I, however, was more severely inhibited by the same mismatches, producing 5-10-fold less ligated product than DNA ligase III (Fig. 3 A).


Figure 3: Reactivity of DNA ligases I and III with DNA substrates containing a single nick with 3` mismatches opposite pyrimidines. The substrates were prepared and assays performed as described under ``Materials and Methods.'' The DNA sequence and structure of the substrate containing a single internal nick with correctly base paired termini is shown on the top of A. Similar versions of this substrate with the indicated mismatch at the 3` terminus of the nick were constructed. In all cases, the top right oligonucleotide (16-mer) was labeled on the 5` end. 3 ng of substrates was used in each reaction. A, joining activity of DNA ligase I with the indicated substrate. Lane 1, no addition; lane 2, 28 fmol; lane 3, 90 fmol; lane 4, 267 fmol; lane 5, 800 fmol of DNA ligase I added. Enzyme concentrations in lanes 6-10 and 11-15 are the same as in lanes 1-5. B, joining activity of DNA ligase III with indicated substrate. Lane 1, no addition; lane 2, 7 fmol; lane 3, 21 fmol; lane 4, 60 fmol; lane 5, 180 fmol of DNA ligase III. Enzyme concentrations in lanes 6-10 and 11-15 are the same as in lanes 1-5. After electrophoresis through a 10% denaturing polyacrylamide gel, labeled oligonucleotides were detected by autoradiography and quantitated by phosphorimage analysis. Although the two DNA ligases have similar specific activities in assays with the oligo(dT)/poly(dA) substrate, DNA ligase III is about 4-fold more active with the control oligonucleotide substrate.



Since the 3`G/T mismatch was more inhibitory than the 3`C/T mismatch, the inhibition may be due to steric effects rather than the absence of correct base pairing. Therefore, we have examined the effects of 3`-mismatched termini opposite purines. DNA joining by DNA ligase I was inhibited more than 50-fold (Fig. 4 A). DNA ligase III was also markedly inhibited by a 3`A/G-mismatched terminus (Fig. 4 B, lanes 7-10), but in contrast with DNA ligase I, the 3`T/G mismatch only reduced DNA joining by 2-fold ( lanes 12-15). The results of assays with DNA ligase II were similar to those shown for DNA ligase III (data not shown). Thus, the inhibition of DNA joining appears to be mediated by steric hindrance, in particular by the 3`-terminal residue. However, DNA ligases II and III are much more tolerant of inappropriate 3` termini than DNA ligase I.


Figure 4: Reactivity of DNA ligases I and III with DNA substrates containing 3` mismatches opposite purines. The substrates were prepared and assays performed as described under ``Materials and Methods.'' The DNA sequence and structure of the substrate containing a single internal nick with correctly base-paired termini is shown on the top of A. Similar versions of this substrate with the indicated mismatch at the 3` terminus of the nick were constructed. In all cases, the top right oligonucleotide (20-mer) was labeled on the 5` end. 3 ng of substrates was used in each reaction. A, joining activity of DNA ligase I with the indicated substrate. Lane 1, no addition; lane 2, 28 fmol; lane 3, 90 fmol; lane 4, 267 fmol; lane 5, 800 fmol of DNA ligase I added. Enzyme concentrations in lanes 6-10 and 11-15 are the same as in lanes 1-5. B, joining activity of DNA ligase III with indicated substrate. Lane 1, no addition; lane 2, 7 fmol; lane 3, 21 fmol; lane 4, 60 fmol; lane 5, 180 fmol of DNA ligase III. Enzyme concentrations in lanes 6-10 and 11-15 are the same as in lanes 1-5. After separation by denaturing gel electrophoresis, the production of a labeled 38-mer by ligation of a 5` P-labeled 20-mer to an 18-mer was detected by autoradiography and quantitated by phosphorimage analysis.



Mammalian DNA Ligase III Is Closely Related to DNA Ligase II and Vaccinia DNA Ligase

A recent peptide mapping study of labeled DNA ligase-adenylate intermediates concluded that the active site regions of DNA ligases II and III are highly related (22) . In an attempt to determine whether these enzymes are derived from the same gene or encoded by separate, homologous genes, we have obtained amino acid sequences from two different preparations of bovine DNA ligase III. After proteolytic digestion and separation of the resultant peptides by reverse phase HPLC, the amino acid sequences of 18 different peptides have been determined. Several peptides isolated from the two different preparations of bovine testis DNA ligase III were identical even though each preparation was purified and cleaved differently (Lys-C digestion of the near-homogenous DNA ligase III from testis nuclei and tryptic digestion of the gel-purified 100-kDa DNA ligase III from testis whole cell extract). A comparison of the 18 peptides with the predicted amino acid sequences of eukaryotic DNA ligases revealed that DNA ligase III exhibits striking homology with vaccinia DNA ligase. Out of the 18 sequences, 13 could be aligned with homologous sequences in vaccinia DNA ligase (Fig. 5). The degree of identity ranged from 30 to 86% with an overall average of 60% for the 177 residues aligned. Several of the DNA ligase III peptides could also be aligned with the catalytic domain of human DNA ligase I, exhibiting about 30% overall identity (data not shown).


Figure 5: Alignment of the peptide sequences from DNA ligases II and III with vaccinia DNA ligase. The peptide sequences from bovine DNA ligase II have been reported previously (18) except for the peptide TQIIQDFLQK. These sequences and peptide sequences from bovine DNA ligase III have been aligned with the predicted amino acid sequence of vaccinia DNA ligase (1-552) (33). A single gap has been introduced for maximum alignment. A hyphen indicates a position within a peptide where it was not possible to assign an amino acid. The 6-residue DNA ligase active site motif (29) is indicated in boldface. The sequence of the DNA ligase II peptide, CAGGHDDATLARLQELDMVK (18), has been modified after reexamination of the amino acid sequencing data and comparison with the homologous DNA ligase III peptide. The COOH-terminal residue of each peptide is underlined. In the absence of unambiguous changes in sequence between homologous peptides from DNA ligase II and III, only amino acids conserved between the peptides and vaccinia DNA ligase are marked with a cross.



As shown previously (18) , peptides derived from homogeneous bovine DNA ligase II also exhibited a similar high degree of identity with vaccinia DNA ligase (Fig. 5). A comparison of DNA ligase II and DNA ligase III peptides that are homologous with vaccinia DNA ligase identified 10 peptides (136 amino acids) with identical sequences (Fig. 5). These peptide sequences encompass almost the entire predicted open reading frame of the vaccinia DNA ligase gene which encodes a 63-kDa polypeptide (33) . Analysis of DNA ligase II and DNA ligase III peptides that were not homologous with vaccinia DNA ligase identified another peptide sequence, Glu-Leu-Tyr-Gln-Leu-Ser-Lys, that was common to both polypeptides, indicating that the homology between DNA ligases II and III extends beyond the bounds of vaccinia DNA ligase.

In the absence of unambiguous changes in amino acid sequence between DNA ligases II and III, it appears that these enzymes are derived from the same gene. We have not detected conversion of 100-kDa DNA ligase III into a 70-kDa active fragment during purification, arguing against nonspecific proteolysis by endogenous proteases. Furthermore, there is no evidence for a liver-specific processing mechanism, since incubation of near homogeneous DNA ligase III with liver nuclear extracts also failed to generate an active 70-kDa fragment (data not shown).

Irrespective of the exact relationship between DNA ligases II and III, it appears that there are two distinct families of eukaryotic DNA ligases, which probably evolved from a common ancestral gene (Fig. 6). One family consists of mammalian DNA ligase I, Drosophila DNA ligase I (35) , S. cerevisiae Cdc9 DNA ligase (36) , and S. pombe Cdc17 DNA ligase (37) . The primary function of these enzymes is to join Okazaki fragments during DNA replication. The second family consists of mammalian DNA ligases II (18) and III, Drosophila DNA ligase II (38) , and the DNA ligases encoded by vaccinia and other pox viruses (33, 39, 40) . These enzymes are probably involved in DNA repair and/or genetic recombination pathways.


DISCUSSION

DNA ligase III has been purified to >90% homogeneity from bovine testis nuclei. We have concluded that the major 100-kDa polypeptide detected by Coomassie Blue staining in the most highly purified fractions is DNA ligase III for the following reasons: (i) the 100-kDa polypeptide cross-reacts with an antiserum raised against a peptide sequence found in all eukaryotic DNA ligases; (ii) the amino acid sequences of peptides derived from the 100-kDa polypeptide exhibit striking homology with the coding sequences of other eukaryotic DNA ligases; (iii) in the presence of labeled ATP, a 100-kDa labeled enzyme-adenylate complex is formed; (iv) in DNA joining assays, the specific activity of DNA ligase III is similar to that of homogeneous DNA ligases I and II.

High levels of both DNA ligase I and DNA ligase III activity were present in whole cell extracts from testes. During spermatogenesis, diploid germ cells replicate their genome to generate a cell with a DNA content of 4 N prior to the two meiotic divisions. Mouse germ cells undergoing premeiotic DNA synthesis contain high levels of DNA ligase I activity (32) , indicating that DNA replication in germ cells is carried out by the same enzymes that function in somatic cells (5, 17) . We suggest that the elevated levels of DNA ligase III reflect the involvement of this enzyme in meiosis. A potential role for DNA ligase III during meiosis would be to complete the large number of homologous recombination events that precede the first meiotic cell division.

In the life cycle of the yeast S. cerevisiae, sporulation is functionally equivalent to gametogenesis in mammals. After transfer to sporulation media, expression of the CDC9 DNA ligase gene, whose product is functionally homologous to mammalian DNA ligase I (28, 34) , is induced prior to the premeiotic S phase (41) . After DNA replication, the cells proceed through the first meiotic division with mature recombinants arising at the end or just after pachytene (42) . Genes in the RAD52 epistasis group were initially isolated, because mutations confer sensitivity to ionizing radiation (43) . Further analysis of these mutants has demonstrated that they are defective in meiosis (44) in addition to DNA strand break repair. The high levels of DNA ligase III in the testes, the association of DNA ligase III with the product of the human strand break repair gene XRCC1 (24) , which is also expressed at high levels in testes (45) , and the decreased levels of DNA ligase III in a xrcc1 mutant cell line EM9, which is defective in DNA strand break repair (24, 46) , are consistent with DNA ligase III also being involved in both meiotic recombination and DNA strand break repair.

The three mammalian DNA ligases were distinguished by their reactivity with different homopolymer substrates (2) . We have investigated the ability of these enzymes to seal nicks with mismatched termini. The substrate specificities of DNA ligases II and III were similar, whereas the substrate specificity of recombinant human DNA ligase I was identical with that of Cdc9 DNA ligase (28) . Thus, the family of functionally homologous replicative DNA ligases appear to be much more sensitive to inhibition by 3` mismatches than the family of DNA ligases that includes DNA ligases II and III and the poxvirus DNA ligases. This may indicate that a stringent enzyme is required to join Okazaki fragments during DNA replication. In contrast, the ability to join nicks with 3`-mismatched termini may be tolerated or preferred in certain DNA repair and recombination pathways.

The differences in amino acid sequence of adenylylated peptides from DNA ligase I and DNA ligase II confirmed that these enzymes are encoded by different genes (18) . The isolation of identical peptides from apparently homogenous preparations of DNA ligases II and III indicates that these enzymes are encoded either by the same gene or by two highly homologous genes. Based on the alignment with vaccinia DNA ligase, the DNA ligase II peptides are distributed over a region of 55 kDa. If we assume that the 70-kDa DNA ligase II consists of 640 amino acids then the 136 amino acids that are identical with DNA ligase III represent 21% of DNA ligase II. Although we cannot exclude the possibility that there are differences in other regions of these polypeptides, the absence of significant differences in amino acid sequence between homologous peptides suggests that these enzymes are probably derived from the same gene. This conclusion is consistent with a recent study which demonstrated that the catalytic domains of DNA ligases II and III are highly related (22) .

We do not believe that DNA ligases II is an active proteolytic fragment of DNA ligase III that is generated by proteolysis during purification for the following reasons: (i) the 70-kDa DNA ligase II polypeptide was blocked to Edman degradation, indicating that it possessed the modified amino-terminal residue of the primary translation product (18) ; (ii) conversion of DNA ligase III to an active fragment similar in size to DNA ligase II has not been observed following incubation of DNA ligase III either with liver nuclear extracts or proteases (2, 22) ; (iii) DNA ligase II and DNA ligase III are present at different levels in different mammalian tissues (2, 18) ; (iv) the mutant Chinese hamster ovary cell line, EM9 has reduced levels of DNA ligase III activity but normal levels of DNA ligase II activity (24, 46, 47) .

Based on similarities in amino acid sequence and/or polynucleotide substrate specificity, the DNA ligases of eukaryotes and eukaryotic viruses can be grouped into two families. This grouping also appears to reflect cellular function. Within the first family, mammalian DNA ligase I, S. cerevisiae Cdc9 DNA ligase and S. pombe Cdc17 DNA ligase have all been shown to be required for DNA replication. The cellular functions of the second family, which consists of mammalian DNA ligases II and III, Drosophila DNA ligase II, and the poxvirus DNA ligases, have been less clearly defined. Vaccinia virus DNA ligase is not required for viral replication, does not affect viral recombination, but influences the sensitivity of the virus to DNA damage (48, 49) . This suggests that vaccinia DNA ligase functions in DNA repair. A similar role has been proposed for DNA ligase II (20, 21) . The high levels of DNA ligase III in the testes, its association with a thymus recombination complex (23) , and its interaction with a DNA strand break repair protein (24, 46) implicate this enzyme in both DNA repair and genetic recombination. We suggest that the DNA ligases in this second family have evolved to fulfill specific functions in pathways of DNA repair and genetic recombination.

In summary, we have purified DNA ligase III to near physical homogeneity from bovine testes. The high level of DNA ligase III in this tissue suggests a role for this enzyme in germ cell development, specifically during meiosis. Amino acid sequencing studies demonstrate that DNA ligase III is highly homologous with vaccinia DNA ligase and appears to be identical with DNA ligase II. The availability of amino acid sequence information from DNA ligase II (18) and DNA ligase III should facilitate the cloning of the gene(s) coding for the two enzymes. This in turn will permit further investigation of their relationship and their respective roles in mammalian DNA metabolism.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grant GM47251 (to A. E. T.) from the Department of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to the experimental work.

To whom correspondence should be addressed. Tel.: 919-990-6173; Fax: 919-990-6147.

The abbreviations used are: FPLC, fast protein liquid chromatography; DTT, dithiothreitol; HPLC, high pressure liquid chromatography; TLCK, N- p-tosyl- L-lysine chloromethyl ketone; TPCK, N-tosyl- L-phenylalanine chloromethyl ketone.


ACKNOWLEDGEMENTS

We thank Dr. Inder Patel for the construction of the recombinant baculovirus that overexpresses human DNA ligase I cDNA.


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