title: In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I.
abstract: Human cDNAs encoding fragments of DNA ligase I, the major replicative DNA ligase in mammalian cells, have been expressed as lacZ fusion proteins in Escherichia coli. A cDNA encoding the carboxyl-terminal catalytic domain of human DNA ligase I was able to complement a conditional-lethal DNA ligase mutation in E. coli as measured by growth of the mutant strain at the non-permissive temperature. Targeted deletions of the amino and carboxyl termini of the catalytic domain identified a minimum size necessary for catalytic function and a maximum size for optimal complementing activity in E. coli. The human cDNA was subjected to systematic site-directed mutagenesis in vitro and mutant polypeptides assayed for functional expression in the E. coli DNA ligase mutant. Such functional analysis of the active site of DNA ligase I identified specific residues required for the formation of an enzyme-adenylate reaction intermediate.
authors.affiliation: Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, UK.
authors.forename: K
authors.initials: K
authors.lastname: Kodama
authors.forename: D E
authors.initials: DE
authors.lastname: Barnes
authors.forename: T
authors.initials: T
authors.lastname: Lindahl
mesh_terms: D000595:Amino Acid Sequence; D001345:Autoradiography; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D004247:DNA / Q000235:genetics*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis
chemical_list: D004277:DNA, Single-Stranded; C000607892:LIG1 protein, human; D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP