title: Characterization of bacteriophage T3 DNA ligase.
abstract: DNA ligases of bacteriophage T4 and T7 have been widely used in molecular biology for decades, but little is known about bacteriophage T3 DNA ligase. Here is the first report on the cloning, expression and biochemical characterization of bacteriophage T3 DNA ligase. The polyhistidine-tagged recombinant T3 DNA ligase was shown to be an ATP-dependent enzyme. The enzymatic activity was not affected by high concentration of monovalent cations up to 1 M, whereas 2 mM ATP could inhibit its activity by 50%. Under optimal conditions (pH 8.0, 0.5 mM ATP, 5 mM DTT, 1 mM Mg(2+) and 300 mM Na(+)), 1 fmol of T3 DNA ligase could achieve 90% ligation of 450 fmol of cohesive dsDNA fragments in 30 min. T3 DNA ligase was shown to be over 5-fold more efficient than T4 DNA ligase for ligation of cohesive DNA fragments, but less active for blunt-ended DNA fragments. Phylogenetic analysis showed that T3 DNA ligase is more closely related to T7 DNA ligase than to T4 DNA ligase.
authors.affiliation: Department of Biochemistry, School of Sciences, Fudan University, Shanghai 200433, P.R. China.
authors.forename: Liang
authors.initials: L
authors.lastname: Cai
authors.forename: Changyun
authors.initials: C
authors.lastname: Hu
authors.forename: Shuiyuan
authors.initials: S
authors.lastname: Shen
authors.forename: Weirong
authors.initials: W
authors.lastname: Wang
authors.forename: Weida
authors.initials: W
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D017903:Bacteriophage T3 / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000494:pharmacology; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000494:pharmacology; D010802:Phylogeny; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D003067:Coenzymes; D007477:Ions; D009711:Nucleotides; D004247:DNA; D011088:DNA Ligases