(Received for publication, December 20, 1995)
From the
The bacteriophage T7 DNA ligase gene was amplified using
polymerase chain reaction-based methods and cloned into a T7
promoter-based expression vector. The protein was overexpressed to
greater than 15% of total soluble protein and purified to homogeneity,
yielding 60-70 mg of protein per liter of bacterial culture. An
initial physical and biochemical characterization of the enzyme reveals
that it exists as a monomer and can ligate nicked, cohesive, and
blunt-ended DNA fragments. Inhibition of the enzyme activity by a
nonhydrolyzable ATP analogue was also investigated. The enzyme has been
crystallized from methoxypolyethylene glycol. The crystals are of the
orthorhombic space group P22
2 and diffract to
2.6 Å. The unit cell dimensions are a = 66.1
Å, b = 87.6 Å, and c = 78.6
Å, with one monomer in the asymmetric unit (V
= 2.77 Å
/Da). This is the first
member of the DNA ligase family of enzymes to be crystallized.
DNA ligases catalyze the formation of phosphodiester bonds at
single-strand breaks between adjacent 3`-hydroxyl and 5`-phosphate
termini in double-stranded DNA (for reviews see (1, 2, 3) ). Polynucleotide ligases are
ubiquitous cell proteins that are required for a number of important
cellular processes, including replication of DNA, and the repair of
damaged DNA, as evidenced by the number of viruses that have genes
encoding there own ligases. Despite their occurrence in all organisms,
DNA ligases show a wide diversity of molecular sizes, amino acid
sequences, and properties. DNA ligases can be divided into two broad
classes: those requiring NAD as a cofactor and those
requiring ATP. The eucaryotic and virally encoded enzymes all require
ATP. The ligases in this class range in size from 103 kDa for the human
type I enzyme (4) to 41 kDa for bacteriophage T7
DNA(5) . The NAD
requiring DNA ligases have
only been found in prokaryotic organisms to date. The amino acid
sequences for a number of bacterial DNA ligases are now
available(6, 7, 8) . These
NAD
-dependent enzymes are highly homologous and are
monomeric proteins of 70-80 kDa but show little homology with
ATP-dependent ligases.
It is now widely accepted that all ligases
catalyze the synthesis of phosphodiester bonds in a very similar
manner, by esterification of a 5`-phosphoryl to a 3`-hydroxyl group.
The reaction mechanism can be split into three distinct catalytic
events (Fig. 1). The first involves activation of the ligase
through the formation of a covalent protein-AMP intermediate. The
nucleotide has been shown to be linked to the enzyme through a
phosphoramidate bond to the -amino group of a conserved active
site lysine(9, 10) . In the second step of the
reaction, the AMP moiety is transferred from the ligase to the
5`-phosphate group at the single-strand break site. Finally, DNA ligase
catalyzes the DNA ligation step with the loss of free AMP. In spite of
these similarities between the two classes of enzyme the manner by
which the bacterial and eucaryotic proteins become activated is rather
different. For eucaryotic ligases, the enzyme-AMP complex is formed
after reaction of the enzyme and ATP with the release of free
pyrophosphate. The bacterial ligases become adenylated in an unusual
reaction, which involves the cleavage of NAD
and the
release of nicotinamide mononucleotide(2) . It has also been
reported that the bacterial enzymes, unlike the ATP-dependent enzymes,
are stimulated up to 20-fold by monovalent cations, particularly
ammonium ions(11) .
Figure 1:
Reaction mechanism of DNA ligases.
Shown is a schematic diagram of the mechanism of action of T7 DNA
ligase. All ATP and NAD-dependent ligases appear to
join DNA in a similar way but utilize a different nucleotide energy
source to catalyze the reaction.
The ATP-dependent DNA ligases contain only a few areas of sequence homology, the most conserved of these is the KXDGXR motif, which has been shown to contain the active site lysine for a number of nucleotidyl transfer enzymes including DNA and RNA ligases (9, 12) and RNA guanylyltransferases (13) . The second most conserved motif (SLRFPRFIRIR) is located in the extreme C termini of the proteins, but its function is currently unknown. Greater homology can be shown if the sequence alignments are limited to more restricted sets(14) . The most highly conserved sequences are located primarily in the C-terminal region of the protein, with the majority of inserts occurring in the N-terminal end.
Bacteriophage T7 encodes a DNA
ligase of molecular mass 41,133 Da based on the gene
sequence(5) . The enzyme can utilize either ATP or, to a lesser
extent, dATP as a cofactor, and catalyzes an exchange reaction between
pyrophosphate and either ATP or dATP. The optimal pH range for the
enzyme is 7.2-7.7(2) . In common with other DNA ligases,
the enzyme also requires a divalent cation for activity. This appears
to be fulfilled by Mg ions in vivo, although
other ions, such as Ca
, can substitute to give
reduced activity. While the 60-kDa DNA ligase from bacteriophage T4 has
been well characterized biochemically and genetically(2) , an
extensive study of the smaller T7 enzyme has not been reported.
Previous work has revealed that both T4 and T7 DNA ligases are able to
join DNA annealed to RNA and, to a slight extent, even RNA annealed to
its complementary RNA strand(15) . Neither enzyme appears to be
capable of ligating single-stranded DNA.
We describe the cloning and overexpression of the T7 DNA ligase gene in Escherichia coli. The gene was placed under the control of a T7 promoter, which allowed us to tightly control the level of gene expression. Strains harboring this plasmid expressed the protein at >15% of soluble cell protein. The enzyme has been purified to near homogeneity, and the physical and biochemical properties of the protein have been evaluated. We have also crystallized the protein using vapor diffusion methods, and these crystals diffract to 2.6 Å.
DNA ligase assay
substrate (22-mer) was radiolabeled by incubating 20 µg of the
oligonucleotide with 100 µCi of [-
P]ATP
(3000 Ci/mmol; Amersham Corp.) and 50 units of T4 polynucleotide kinase
for 45 min at 37 °C followed by 10 min at 70 °C. The
unincorporated label was removed by centrifugation through a S-200
microspin column (Pharmacia). The DNA ligase assay was performed
essentially as described previously(23) . The complementary 18-
and 22-mer oligonucleotides were annealed to single-stranded M13mp19 by
incubation at 70 °C for 2 min and allowed to cool for 1 h. The
annealed DNA was incubated with ligase buffer (50 mM Tris, pH
7.5, 10 mM MgCl
, 5 mM DTT) unless
otherwise stated, in the presence of enzyme and nucleotide cofactors as
indicated, in a total volume of 10 µl for 15 min at 25 °C. The
reactions were terminated by the addition of sequencing stopping buffer
(Sequenase kit, U.S. Biochemical Corp.) followed by heating at 95
°C for 5 min. The ligation products were subjected to
electrophoresis on a 15% polyacrylamide urea gel and to autoradiography
with Fugi RX x-ray film.
Ligation of blunt-ended DNA fragments was
performed in ligase buffer plus 1 mM ATP, with 200 ng of HaeIII-digested X174 DNA, polyethylene glycol 8000 and 2
units of ligase. Reactions were incubated for 30 min at 25 °C, and
products were analyzed on 1% agarose gels.
The T7 DNA
ligase gene was fully sequenced, and the sequence agreed with that
published previously(5) . The resulting active clone was
screened for overexpression of the T7 ligase gene by transforming into
B834(DE3)[pLysS], inoculating single colonies into 5-ml
cultures of LB containing ampicillin and chloramphenicol and growing
for several hours. The cells were induced by the addition of
isopropyl-1-thio--D-galactopyranoside and grown for 3 h.
Samples were taken and analyzed by SDS-PAGE. In all cases a large
amount of a protein with an apparent molecular mass of approximately 40
kDa accumulated in the induced cells but was absent from uninduced
cultures. The crude sonicated cell extract supernatant was precipitated
with ammonium sulfate. The protein was then purified using a
heparin-Sepharose column followed by blue-Sepharose, Q-Sepharose, and
finally gel filtration using a Superdex S-200 column. The protein was
greater than 99% pure at this stage as determined by 12% SDS-PAGE (Fig. 2). The final yield of enzyme was typically 60-70
mg/liter using the host strain B834(DE3) compared with 10-15
mg/liter when BL21(DE3) was used as the expression host(26) .
Figure 2: Purification of T7 DNA ligase. A 12% SDS-polyacrylamide gel showing the level of expression of T7 ligase and the subsequent purification steps. Lane 1, uninduced B834(DE3)[pLysS] (pT7lig); lane 2, induced B834(DE3)[pLysS] (pT7lig); lane 3, ammonium sulfate precipitated cell extract; lane 4, heparin-Sepharose purification; lane 5, blue-Sepharose purification; lane 6, fast protein liquid chromatography mono-Q purification; lane 7, low molecular weight markers (Sigma Dalton VII).
Figure 3: Analytical gel filtration of T7 ligase. The elution profile of T7 ligase from a Superdex S-200 gel filtration column. The column was calibrated by monitoring the elution of standard proteins at 280 nm. A 100-µl sample of purified T7 ligase (2 mg/ml) was loaded onto the column (pre-equilibrated in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA). Fractions of 1 ml were collected, and peak fractions were analyzed by SDS-PAGE.
Mass spectroscopy and equilibrium sedimentation were employed to determine whether the enzyme exists as a monomer or dimer. According to equilibrium sedimentation, the protein appears to be monomeric with a calculated molecular weight of 40,645 ± 300 (Fig. 4). The molecular mass of native T7 ligase was determined to be 41,132 Da using electrospray mass spectroscopy (Fig. 4), with the adenylated form having a mass of 41,460 Da. Approximately one-third of the purified protein exists in the adenylated form.
Figure 4: Physical properties of T7 ligase. A, analytical equilibrium centrification. Sedimentation equilibrium of T7 ligase at 15,000 rpm and a constant temperature of 20 °C. The data are consistent with a protein of molecular weight 40,625 ± 262. Mass spectroscopy. Electrospray mass spectroscopy reveals that the purified T7 DNA ligase is composed of two major species, the native enzyme with a molecular mass of 41,132 Da and the adenylated enzyme with a molecular mass of 41,460 Da.
The isoelectric point (pI) of T7 ligase has been calculated to be 5.2 based on the amino acid sequence. The isoelectric point of purified T7 ligase was determined using a PhastGel (Pharmacia). The pI was shown to be close to 5.8 (data not shown), but the protein is insoluble below its pI.
Figure 5:
DNA
ligase assay. The synthetic nicked substrate consists of a
complementary and 5`-P-labeled 22-mer and an adjacent
18-mer annealed with single-stranded M13 DNA. Reactions containing DNA
substrate and 1 unit of either T7 or T4 DNA ligase were incubated for
15 min at room temperature in ligation buffer (see ``Experimental
Procedures'') with other additions as indicated. Panels A and B show the autoradiographs of electrophoresis
products of either T7 (A) or T4 (B) ligases,
respectively, separated on 15% denaturing polyacrylamide gels. Lane
1, no ATP; lane 2, 5 mM ATP; lane 3, 5
mM AMPPNP; lane 4, 10 mM EDTA; lane
5, 5 mM PP
; lane 6, control with
single-stranded M13 DNA omitted; lane 7, control with no
ligase.
Figure 6:
Blunt-ended DNA ligase assay. Ligase
reactions were performed with 0.5 µg of HaeIII-digested
X174. PEG 6000 was incubated with HaeIII DNA fragments
at the final percentage indicated below and then ligated with 0.5 units
of T7 or T4 DNA ligase. The reactions were terminated after 30 min by
the addition of EDTA and heating at 65 °C. Lanes A and P, EcoRI/HindIII-digested
DNA
molecular weight markers; lanes B-H, 0, 5, 10, 1, 20, 25
and 30% PEG with 0.5 units of T7 ligase; lanes I-O, 0,
5, 10, 15, 20, 25, and 30% PEG with 0.5 units of T4
ligase.
It has
been shown that T7 ligase requires magnesium and uses ATP, and to a
lesser extent dATP, as the cofactor in the DNA joining reaction (2) . The K for ATP or dATP in the joining
reaction is approximately 6
10
M.
In the exchange reaction the K
for ATP is 3
10
M, while that for dATP is
10-fold higher(2) . We have examined the catalytic properties
of the enzyme using a radiolabeled oligonucleotide assay described
previously(23) . The DNA substrate consists of two
oligonucleotide primers (18-mer and 22-mer), one 5`-labeled with
[
-
P]ATP, annealed adjacently onto
single-stranded M13 DNA, creating a single synthetic nick site. We
observed that ligation of this nicked substrate and cohesive end DNA
fragments by both T7 and T4 ligases occurs at comparable rates,
agreeing with the published values (30) of approximately 1,200
Weiss units/mg of protein (data not shown). We observed that both
purified T7 and commercial T4 DNA ligases can efficiently ligate DNA in
the absence of ATP (Fig. 5). Mass spectroscopy analysis of the
purified protein revealed that greater than 30% of the T7 enzyme exists
in the adenylated form (Fig. 4). This adduct is very stable to
acid and alkali treatments, which has also been reported for other DNA
ligases(31) . Many reports have shown that AMP can be removed
by preincubating DNA ligase with either nicked DNA or
PP
(31, 32) . However, we were unable to
reduce the level of adenylation of the T7 enzyme to less than 10% after
these treatments. The addition of PP
caused complete
inhibition of ligation by adenylated T7 ligase in the absence of ATP
but was less effective on the T4 enzyme under similar conditions (Fig. 5). It has been shown previously that incubating T4
ligase-AMP with PP
results in the release of the adenylate
moiety and the appearance of ATP(31) . However, it is possible
to restore activity of the T7 enzyme by removal of PP
by
dialysis even in the presence of activated charcoal. Enzyme treated in
this way retained the ability to perform ligations in the absence of
ATP, and thus a significant proportion of the enzyme must still be in
the adenylated form.
The addition of 5 mM EDTA inhibits completely the formation of the AMP adduct (data not shown). We found that adenylated T7 and T4 ligases can perform the AMP transfer and sealing steps even in the presence of 10 mM EDTA (Fig. 5). This contrasts with reports of an absolute requirement for divalent metal ions in the transfer and sealing reactions of human I, T4, and E. coli ligases(23, 33, 34) .
A variety of
nucleotides (CTP, dTTP, GTP, NAD) were unable to
substitute for ATP in the ligation reaction and also did not inhibit
the adenylated enzyme. They also had no effect on the reaction in the
presence of ATP, even at concentrations up to 5 mM (data not
shown). However, the ATP analogue, AMPPNP, inhibited significantly the
ligation of nicked (Fig. 5) and cohesive end DNA (data not
shown) by the adenylated T7 enzyme in the absence of ATP. This ATP
analogue contains a bridging nitrogen instead of oxygen between the
- and
-phosphates. It is interesting that this modification
makes the nucleotide an inhibitor (Fig. 5), since all DNA
ligases characterized to date cleave ATP between the
- and
-phosphates releasing PP
(Fig. 1). In contrast,
adenylated T4 DNA ligase is not inhibited by AMPPNP under similar
conditions (Fig. 5), nor does AMPPNP affect the activity of T4
ligase in the presence of ATP. To characterize further the inhibitory
effects of AMPPNP on T7 ligase, increasing amounts of the analogue were
incubated with the enzyme prior to the addition of DNA substrate. These
results showed that a decrease in ligation activity was directly
proportional to the concentration of AMPPNP (data not shown). Levels in
excess of 10 mM were required to inhibit the enzyme by greater
than 90%.
The T7 enzyme was incubated with either
[-
P]ATP or
[
-
P]ATP in ligase buffer as described
previously (35) . The protein was separated by SDS-PAGE, and
autoradiography showed that the enzyme had become selectively labeled
with the
- but not the
-labeled ATP substrate (Fig. 7), in common with other DNA ligases.
Figure 7:
Adenylation of T7 DNA ligase. T7 ligase
was incubated with either [-
P]ATP (lane
1) or [
-
P]ATP (lane 2), and lane 3 contains labeled ATP only, in the presence of 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 5 mM MgCl
. The samples were separated on a 15%
SDS-polyacrylamide gel and exposed to
autoradiography.
Figure 8:
T7 DNA ligase crystals. Crystals of T7
ligase were grown using hanging drops. They belong to the space group
P22
2 and grew to a maximum size of 0.8
0.5
0.2 mm.
The crystals were only moderately stable on exposure to
x-rays but it was possible to collect complete native data to 2.8
Å from a number of crystals using synchrotron radiation
(Daresbury, UK; EMBL, Hamburg, Germany; and Brookhaven National
Laboratory, Upton, NY) and an image plate detector. The crystals are
orthorhombic and belong to the space group P22
2
with unit cell dimensions of a = 66.1 Å, b = 87.6 Å, c = 78.6 Å. Assuming
that there is one monomer in the asymmetric unit, these crystals have a
calculated V
of 2.77
Å
/Da(36) . Crystal lifetime has been extended
further by flash freezing at 100 K in the presence of 25% glycerol, and
this has allowed 2.6 Å data to be collected from a single
crystal. Selenomethionine-substituted protein has also been
crystallized, and data have been collected to 2.8-Å resolution.
The selenium positions have been determined using direct methods as
implemented in SHELX (37) and used to estimate an initial set
of phases. These phases have been used to determine heavy atom
positions in a number of other derivatives using difference Fourier
techniques. The resulting electron density map is of sufficient quality
to reveal secondary structural elements, and model building is under
way.
A number of the larger eucaryotic/viral ATP-dependent ligases have been studied in detail at the genetic and biochemical level. In this report we describe the characterization of bacteriophage T7 DNA ligase, one of the smallest members of this family of enzymes. We have cloned and overexpressed T7 DNA ligase in E. coli and purified the protein in large amounts, allowing us to characterize some of its physical and biochemical properties. We have confirmed the previously deduced molecular mass of the native enzyme to be 41,133 Da using mass spectroscopy. This analysis also revealed that approximately 30% of the purified enzyme exists in the adenylated form, in common with a number of other ligases(7) . The adenylated form of the enzyme is remarkably stable, and the adduct is resistant to acid and alkali treatments. The discrepancy in the molecular weight determined by gel filtration and mass spectroscopy indicates an irregular shape for the ligase molecule, which has been suggested for other DNA ligases on the basis of analytical gel filtration and sedimentation centrifugation data(38, 39) . Mammalian DNA ligase I has been shown to have a markedly asymmetric structure with a frictional ratio of 1.9 (40) and resembles the E. coli and T4 DNA ligases in this regard. Preliminary examination of the molecular boundary in the initial electron density maps reveals that this is also the case for T7 ligase (data not shown).
T7 ligase has a DNA substrate profile that
is similar to the T4 DNA ligase. It can join cohesive ends at a rate
similar to that of the T4 enzyme, but it is less efficient at
blunt-ended DNA ligations, with little activity being observed in the
absence of PEG. Activity of the adenylated enzyme is inhibited by
PP and AMPPNP. These compounds presumably inhibit the
enzyme either by preventing the transfer of the AMP to the 5`-phosphate
of the DNA or by inhibition of the final sealing step of the reaction.
It is likely that PP
and AMPPNP are binding in or near to
the active site pocket, since inhibition appears to be competitive and
is reversible. The binding of these compounds may inhibit the enzyme by
interfering with the nucleotidyl transfer to the DNA nick site and/or
the sealing reactions, perhaps by displacing the AMP moiety.
Interestingly, adenylated T7 ligase was not inhibited even by very high
concentrations of ATP (up to 10 mM), even though similar
levels of AMPPNP inhibited the enzyme significantly. The reason for
this difference is not clear at present. However, it has been shown,
for both human DNA ligase I and II, that ATP can inhibit the final step
in the ligation reaction, which involves the release of AMP from the
adenylated DNA intermediate(23) . It is suggested that this
inhibition may be due to competition for the AMP-binding site in DNA
ligase between the AMP moiety in the AMP-DNA complex and ATP. These
workers have demonstrated also that another ATP analogue, F-ara-ATP, is
a potent inhibitor of human I ligase activity(41) . This
analogue appears to compete with ATP and thus inhibits formation of the
ligase-AMP adduct. However, a second possibility is that there is a
second nucleotide binding site that may be the target for inhibition.
We cannot distinguish between these two models with the present data.
The crystallization and characterization of T7 DNA ligase will have important implications for the understanding the structure and function of this evolutionary conserved class of DNA modifying enzymes and related proteins involved in nucleotidyl transfer reactions. Shuman and co-workers (42) have predicted that ligases and eucaryotic RNA capping enzymes share a common mechanism of covalent catalysis. These capping enzymes contain a similar active site motif to DNA ligase (KXDG), and replacement of the active site lysine with alanine was shown to be lethal(43) , in common with a number of RNA and DNA ligases(10, 12) . A closer comparison of the sequences of DNA ligases and RNA capping enzymes suggested that these enzymes share four to five motifs, which are arranged in the same order and with similar spacing between them(13) . Mutation analysis of these motifs in the capping enzyme CEG1 from Saccharomyces cerevisiae has shown that these conserved sequences play important roles in these enzymes (13) . The conservation of these essential motifs in polynucleotide ligases and capping enzymes may reflect a common ancestry, as both types of enzymes catalyze single nucleotidyl transfer reactions to the ends of polynucleotide chains. The determination of the crystal structure of T7 ligase should provide some insight into the structural basis of nucleotidyl transfer and ligation reactions.