Characterization of a temperature-sensitive DNA ligase from Escherichia coli

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 degreesC purified LigA251 has 20-fold lower ligation activity in vitro, and its activity is reduced further at 42 degreesC, 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.