From the
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.
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.
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 H
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
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.
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.
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.
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.
We thank Dr. Inder Patel for the construction of the
recombinant baculovirus that overexpresses human DNA ligase I cDNA.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
0. 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.
-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.
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.
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) .
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.
- p-tosyl-
L-lysine
chloromethyl ketone; TPCK, N-tosyl-
L-phenylalanine
chloromethyl ketone.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.