Bacteriophage T7 DNA Ligase

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 P21212 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 (Vm = 2.77 Å3/Da). This is the first member of the DNA ligase family of enzymes to be crystallized. 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 P21212 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 (Vm = 2.77 Å3/Da). This is the first member of the DNA ligase family of enzymes to be crystallized. INTRODUCTIONDNA 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.Lehman I.R. Science. 1974; 186: 790Crossref PubMed Scopus (468) Google Scholar, 2.Engler M.J. Richardson C.C. Boyer P.D. The Enzymes. XV. Academic Press, Inc., New York1982: 3-29Google Scholar, 3.Lindahl T. Barnes D.E. Annu. Rev. Biochem. 1992; 61: 251-281Crossref PubMed Scopus (187) Google Scholar)). 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.Barnes D.E. Johnson L.H. Kodama K. Tomkinson A.E. Lasko D.D. Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6679-6683Crossref PubMed Scopus (125) Google Scholar) to 41 kDa for bacteriophage T7 DNA(5.Dunn J.J. Studier F.W. J. Mol. Biol. 1981; 148: 303-330Crossref PubMed Scopus (126) Google Scholar). 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.Ishino Y. Shinagawa H. Makino K. Tsunasawa S. Sakiyama F. Nakata A. Mol. & Gen. Genet. 1986; 204: 1-7Crossref PubMed Scopus (43) Google Scholar, 7.Barany F. Gelfand D.H. Gene (Amst.). 1991; 109: 1-11Crossref PubMed Scopus (73) Google Scholar, 8.Lauer G. Rudd E.A. McKay D.L. Ally A. Ally D. Backman K.C. J. Bacteriol. 1991; 173: 5047-5053Crossref PubMed Google Scholar). 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 ϵ-amino group of a conserved active site lysine(9.Tomkinson A.E. Totty N.F. Ginsburg M. Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 400-404Crossref PubMed Scopus (106) Google Scholar, 10.Kodama K. Barnes D.E. Lindahl T. Nucleic Acids Res. 1991; 19: 6093-6099Crossref PubMed Scopus (81) Google Scholar). 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.Engler M.J. Richardson C.C. Boyer P.D. The Enzymes. XV. Academic Press, Inc., New York1982: 3-29Google Scholar). 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.Modrich P. Lehman I.R. J. Biol. Chem. 1973; 248: 7502-7511Abstract Full Text PDF PubMed Google Scholar).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.Tomkinson A.E. Totty N.F. Ginsburg M. Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 400-404Crossref PubMed Scopus (106) Google Scholar, 12.Heaphy S. Singh M. Gait M.J. Biochemistry. 1987; 26: 1688-1696Crossref PubMed Scopus (48) Google Scholar) and RNA guanylyltransferases (13.Shuman S. Liu Y. Schwer Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The second most conserved is in the termini of the but is homology can be shown the sequence are to A. Nucleic Acids Res. 1992; PubMed Scopus Google Scholar). The most highly conserved sequences are in the of the with the of in the T7 a DNA ligase of molecular the gene J.J. Studier F.W. J. Mol. Biol. 1981; 148: 303-330Crossref PubMed Scopus (126) Google Scholar). The enzyme can ATP to a as a and catalyzes an reaction between and ATP The range for the enzyme is M.J. Richardson C.C. Boyer P.D. The Enzymes. XV. Academic Press, Inc., New York1982: 3-29Google Scholar). In with DNA ligases, the enzyme also a for This to be by in as can to the DNA ligase from bacteriophage has been and M.J. Richardson C.C. Boyer P.D. The Enzymes. XV. Academic Press, Inc., New York1982: 3-29Google an of the T7 enzyme has been has that and T7 DNA ligases are to DNA to RNA to a RNA to RNA Richardson C.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). enzyme to be of the and of the T7 DNA ligase gene in The gene was the of a T7 which to the of gene this the protein at of soluble cell The enzyme has been purified to homogeneity, and the physical and biochemical of the protein have been have also crystallized the protein using and these crystals diffract to 2.6 and of as for 1987; Scholar) for the of and the of DNA for J.J. J. Bacteriol. PubMed Google a DNA ligase to the ligase and gene J. Mol. Biol. PubMed Scopus Google Scholar, F.W. J. Mol. Biol. 1986; PubMed Scopus Google Scholar) for the of T7 DNA in the and enzymes to the the DNA and as J. T. Press, Scholar). using an DNA The to the chain chain of the T7 the T7 sequence a an and a a was with these and bacteriophage T7 DNA using in of and as Gelfand D.H. S. Science. PubMed Scopus Google Scholar). The reaction was in and the was from the and purified using a DNA and of the T7 was purified as and with and into and into the ligase gene by and for expression by into at in of and to an of with and for a The of was using F. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) was to the nucleotide sequence of the T7 DNA was in using to the of T7 of and with a of and at the The by the of was for a of the by at The cell at The by of a cell in and of the cell by at the was by the of an of a ammonium The was by at and the was in a of which in a was than that of a of This was to a with and the with a of this the ligase was by with that this the enzyme at The protein was with to the to and this was a which been in A. The protein was with The of in the was to than by with A. This was a in A. The was with of of the protein with was by a by a of DNA with was in a of of DNA, and nucleotide as at for and by the of by at for in and the was with ligase was by of the with of and of for at by at The was by through a The DNA ligase was as J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). The and to by at for and to for The DNA was with ligase in the of enzyme and nucleotide as in a total of for at The by the of by at for The ligation to a 15% and to with of blunt-ended DNA was in ligase with of DNA, and of for at and DNA ligase was in and of ATP in a total of for at The by in for and by a PubMed Scopus Google Scholar). The protein was to between and using The protein was into and this by at a in a of and methoxypolyethylene of molecular at a of have been to 2.6 using and and an The using S. and Scholar) and with and of T7 the enzyme in it was to by the T7 gene from bacteriophage T7 DNA and it into a T7 promoter-based expression vector. The an the to be cloned into The was to to at this The a site after the The was to and the DNA was and The DNA was with and and This was into into and number of found to have the DNA size with and a of the to a DNA ligase that is at the that the T7 ligase gene is the of a T7 RNA polymerase which be in this cell of expression of the T7 ligase gene be that is to the as a of from the the which is the DNA as the sequence of the ligase T7 DNA ligase gene was and the sequence with that J.J. Studier F.W. J. Mol. Biol. 1981; 148: 303-330Crossref PubMed Scopus (126) Google Scholar). The active was for of the T7 ligase gene by into into of and and for The by the of and for and by In all a of a protein with an molecular of kDa in the but was from The cell was with ammonium The protein was purified using a by and using a The protein was greater than at this as by (Fig. The of enzyme was 60-70 using the with was as the expression Nucleic Acids Res. PubMed Scopus Google of T7 DNA the of expression of T7 ligase and the ammonium cell protein molecular of T7 DNA ligase has a molecular of amino acid the it was that the protein in the of a that the protein was that it was to this T7 ligase was a that been with proteins of molecular These in the of in the The protein from the with an molecular of kDa (Fig. that this the molecular for T7 ligase the of a number of the and the of the protein to In the of and the this very at of the This was first in the of to of protein that was by The mechanism by which this is but be the of the enzyme has been of T7 The of T7 ligase from a The was by the of proteins at of purified T7 ligase was the in of and by and to the enzyme exists as a monomer to the protein to be monomeric with a molecular of (Fig. The molecular of T7 ligase was to be using (Fig. with the adenylated a of of the purified protein exists in the adenylated of T7 of T7 ligase at and a of The are with a protein of molecular reveals that the purified T7 DNA ligase is of two the enzyme with a molecular of and the adenylated enzyme with a molecular of of T7 ligase has been to be the amino acid The of purified T7 ligase was using a The was shown to be to but the protein is and of T7 of purified T7 ligase was a of and DNA It was shown that T7 DNA in can ligate and Richardson C.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The purified enzyme is very at DNA and (Fig. it is of single-strand DNA in with the and ligases (Fig. DNA ligase has the to blunt-ended DNA Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and this activity is by the of as Nucleic Acids Res. PubMed Scopus Google Scholar). The of ligases, as those from and to Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). T7 ligase can ligate blunt-ended DNA in the of a wide range of but is in the of This activity is at between and of and the of the reaction these is with that of the enzyme (Fig. ligase The of a and and an adjacent with DNA and unit of T7 DNA ligase for at in ligation with as and show the of of T7 ligases, 15% with DNA with DNA ligase with of was with DNA at the and with of T7 DNA The after by the of and at and DNA molecular and with of T7 and with of has been shown that T7 ligase and and to a as the cofactor in the DNA reaction M.J. Richardson C.C. Boyer P.D. The Enzymes. XV. Academic Press, Inc., New York1982: 3-29Google Scholar). The for ATP in the reaction is In the reaction the for ATP is that for is M.J. Richardson C.C. Boyer P.D. The Enzymes. XV. Academic Press, Inc., New York1982: 3-29Google Scholar). have the catalytic of the enzyme using a J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). The DNA of two and one with DNA, a site. that ligation of this and DNA by T7 and ligases at with the Richardson C.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar) of of protein that purified T7 and DNA ligases can ligate DNA in the of ATP (Fig. of the purified protein that greater than of the T7 enzyme exists in the adenylated (Fig. This is very to acid and which has also been reported for DNA A. Richardson C.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). have shown that AMP can be by DNA ligase with DNA A. Richardson C.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). to the of of the T7 enzyme to than after these The of of ligation by adenylated T7 ligase in the of ATP but was the enzyme similar (Fig. It has been shown that with in the release of the moiety and the of A. Richardson C.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). it is to activity of the T7 enzyme by of by in the of activated in this the to in the of and a of the enzyme be in the adenylated of the formation of the AMP found that adenylated T7 and ligases can the AMP transfer and in the of (Fig. This with of an for in the transfer and of human and J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 61: PubMed Scopus (48) Google Scholar, Richardson C.C. J. Biol. Chem. Full Text PDF PubMed Google of to for ATP in the ligation reaction and also the adenylated also the reaction in the of at up to the ATP the ligation of (Fig. and DNA by the adenylated T7 enzyme in the of ATP. This ATP analogue a of between the and It is that this the nucleotide an (Fig. all DNA ligases to ATP between the and (Fig. 1). In adenylated DNA ligase is by similar (Fig. the activity of ligase in the of ATP. the of T7 of the analogue with the enzyme to the of DNA These that a in ligation activity was to the of in of required to the enzyme by greater than T7 enzyme was with in ligase as 1986; Scopus Google Scholar). The protein was by and that the enzyme become with the but the ATP (Fig. in with DNA of T7 DNA T7 ligase was with and ATP in the of The a 15% and to ligase crystals as which after and a size of (Fig. in the of the crystals this in to in that been for and size after This was also to of crystals to and to DNA ligase of T7 ligase using to the space group P21212 and to a size of crystals only to but it was to to from a number of crystals using and and an The crystals are orthorhombic and to the space group P21212 with unit cell dimensions of a = 66.1 Å, b = 87.6 Å, c = 78.6 Å. that there is one monomer in the asymmetric these crystals have a of 2.77 J. Mol. Biol. PubMed Scopus Google Scholar). has been by at in the of and this has 2.6 to be from a protein has also been and have been to The have been using methods as in G. D. Press, Scholar) and to an initial of These have been to in a number of using The is of to and is number of the ATP-dependent ligases have been in at the and biochemical In this the characterization of bacteriophage T7 DNA one of the of this family of have cloned and overexpressed T7 DNA ligase in and purified the protein in to of physical and biochemical properties. have the molecular of the enzyme to be using This also that of the purified enzyme exists in the adenylated in with a number of F. Gelfand D.H. Gene (Amst.). 1991; 109: 1-11Crossref PubMed Scopus (73) Google Scholar). The adenylated of the enzyme is and the is to acid and The in the molecular by and an for the ligase which has been for DNA ligases the of and P. Y. Lehman I.R. J. Biol. Chem. 1973; 248: Full Text PDF PubMed Google Scholar, A. K. Biochemistry. 1973; PubMed Scopus Google Scholar). DNA ligase I has been shown to have a asymmetric with a of A.E. Lasko D.D. G. Lindahl T. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar) and the and DNA ligases in this of the molecular in the initial reveals that this is also the for T7 ligase ligase has a DNA that is similar to the DNA It can at a similar to that of the but it is at blunt-ended DNA with little activity in the of of the adenylated enzyme is by and These the enzyme by the transfer of the AMP to the 5′-phosphate of the DNA by of the step of the It is that and are in to the active site to be and is The of these the enzyme by with the nucleotidyl transfer to the DNA site the by the AMP adenylated T7 ligase was by very of ATP to similar of the enzyme The for this is at it has been for human DNA ligase I and that ATP can the step in the ligation reaction, which involves the release of AMP from the adenylated DNA J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). It is that this be to for the site in DNA ligase between the AMP moiety in the complex and ATP. These have also that ATP is a of human I ligase P. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). This analogue to with ATP and formation of the a second is that there is a second nucleotide site that be the for between these two with the and characterization of T7 DNA ligase have important for the the and of this conserved class of DNA enzymes and proteins in nucleotidyl transfer and S. M. H. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) have that ligases and eucaryotic RNA enzymes a mechanism of covalent These enzymes contain a similar active site to DNA ligase and of the active site lysine with was shown to be S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google in with a number of RNA and DNA K. Barnes D.E. Lindahl T. Nucleic Acids Res. 1991; 19: 6093-6099Crossref PubMed Scopus (81) Google Scholar, 12.Heaphy S. Singh M. Gait M.J. Biochemistry. 1987; 26: 1688-1696Crossref PubMed Scopus (48) Google Scholar). of the sequences of DNA ligases and RNA enzymes that these enzymes to which are in the and with similar between S. Liu Y. Schwer Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of these in the enzyme from has shown that these conserved sequences important in these enzymes (13.Shuman S. Liu Y. Schwer Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of these in ligases and enzymes a as of enzymes catalyze nucleotidyl transfer to the of The of the of T7 ligase into the of nucleotidyl transfer and ligation INTRODUCTIONDNA 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.Lehman I.R. Science. 1974; 186: 790Crossref PubMed Scopus (468) Google Scholar, 2.Engler M.J. Richardson C.C. Boyer P.D. The Enzymes. XV. Academic Press, Inc., New York1982: 3-29Google Scholar, 3.Lindahl T. Barnes D.E. Annu. Rev. Biochem. 1992; 61: 251-281Crossref PubMed Scopus (187) Google Scholar)). 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.Barnes D.E. Johnson L.H. Kodama K. Tomkinson A.E. Lasko D.D. Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6679-6683Crossref PubMed Scopus (125) Google Scholar) to 41 kDa for bacteriophage T7 DNA(5.Dunn J.J. Studier F.W. J. Mol. Biol. 1981; 148: 303-330Crossref PubMed Scopus (126) Google Scholar). 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.Ishino Y. Shinagawa H. Makino K. Tsunasawa S. Sakiyama F. Nakata A. Mol. & Gen. Genet. 1986; 204: 1-7Crossref PubMed Scopus (43) Google Scholar, 7.Barany F. Gelfand D.H. Gene (Amst.). 1991; 109: 1-11Crossref PubMed Scopus (73) Google Scholar, 8.Lauer G. Rudd E.A. McKay D.L. Ally A. Ally D. Backman K.C. J. Bacteriol. 1991; 173: 5047-5053Crossref PubMed Google Scholar). 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 ϵ-amino group of a conserved active site lysine(9.Tomkinson A.E. Totty N.F. Ginsburg M. Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 400-404Crossref PubMed Scopus (106) Google Scholar, 10.Kodama K. Barnes D.E. Lindahl T. Nucleic Acids Res. 1991; 19: 6093-6099Crossref PubMed Scopus (81) Google Scholar). 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.Engler M.J. Richardson C.C. Boyer P.D. The Enzymes. XV. Academic Press, Inc., New York1982: 3-29Google Scholar). 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.Modrich P. Lehman I.R. J. Biol. Chem. 1973; 248: 7502-7511Abstract Full Text PDF PubMed Google Scholar).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.Tomkinson A.E. Totty N.F. Ginsburg M. Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 400-404Crossref PubMed Scopus (106) Google Scholar, 12.Heaphy S. Singh M. Gait M.J. Biochemistry. 1987; 26: 1688-1696Crossref PubMed Scopus (48) Google Scholar) and RNA guanylyltransferases (13.Shuman S. Liu Y. Schwer Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The second most conserved is in the termini of the but is homology can be shown the sequence are to A. Nucleic Acids Res. 1992; PubMed Scopus Google Scholar). The most highly conserved sequences are in the of the with the of in the T7 a DNA ligase of molecular the gene J.J. Studier F.W. J. Mol. Biol. 1981; 148: 303-330Crossref PubMed Scopus (126) Google Scholar). The enzyme can ATP to a as a and catalyzes an reaction between and ATP The range for the enzyme is M.J. Richardson C.C. Boyer P.D. The Enzymes. XV. Academic Press, Inc., New York1982: 3-29Google Scholar). In with DNA ligases, the enzyme also a for This to be by in as can to the DNA ligase from bacteriophage has been and M.J. Richardson C.C. Boyer P.D. The Enzymes. XV. Academic Press, Inc., New York1982: 3-29Google an of the T7 enzyme has been has that and T7 DNA ligases are to DNA to RNA to a RNA to RNA Richardson C.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). enzyme to be of the and of the T7 DNA ligase gene in The gene was the of a T7 which to the of gene this the protein at of soluble cell The enzyme has been purified to homogeneity, and the physical and biochemical of the protein have been have also crystallized the protein using and these crystals diffract to 2.6 Å.