title: Characterization of a temperature-sensitive DNA ligase from Escherichia coli.
abstract: DNA ligases are essential enzymes in cells due to their ability to join DNA strand breaks formed during DNA replication. Several temperature-sensitive mutant strains of Escherichia coli, including strain GR501, have been described which can be complemented by functional DNA ligases. Here, it is shown that the ligA251 mutation in E. coli GR501 strain is a cytosine to thymine transition at base 43, which results in a substitution of leucine by phenylalanine at residue 15. The protein product of this gene (LigA251) is accumulated to a similar level at permissive and non-permissive temperatures. Compared to wild-type LigA, at 20 degrees C purified LigA251 has 20-fold lower ligation activity in vitro, and its activity is reduced further at 42 degrees C, resulting in 60-fold lower ligation activity than wild-type LigA. It is proposed that the mutation in LigA251 affects the structure of the N-terminal region of LigA. The resulting decrease in DNA ligase activity at the non-permissive temperature is likely to occur as the result of a conformational change that reduces the rate of adenylation of the ligase.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
authors.forename: Manuel
authors.initials: M
authors.lastname: Lavesa-Curto
authors.forename: Heather
authors.initials: H
authors.lastname: Sayer
authors.forename: Desmond
authors.initials: D
authors.lastname: Bullard
authors.forename: Andrew
authors.initials: A
authors.lastname: MacDonald
authors.forename: Adam
authors.initials: A
authors.lastname: Wilkinson
authors.forename: Andrew
authors.initials: A
authors.lastname: Smith
authors.forename: Laura
authors.initials: L
authors.lastname: Bowater
authors.forename: Andrew
authors.initials: A
authors.lastname: Hemmings
authors.forename: Richard P
authors.initials: RP
authors.lastname: Bowater
mesh_terms: D000595:Amino Acid Sequence; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D011088:DNA Ligases; C120640:DNA ligase (NAD); C497118:LigA protein, E coli