Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA).
NAD(+)-dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD(+)-dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo.
Wilkinson
Adam
A
Phico Therapeutics Ltd, Babraham Hall, Babraham, Cambridge, CB2 4AT, UK.
Smith
Andrew
A
Bullard
Desmond
D
Lavesa-Curto
Manuel
M
Sayer
Heather
H
Bonner
Alexandra
A
Hemmings
Andrew
A
Bowater
Richard
R
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009243:NAD; D004247:DNA; D011088:DNA Ligases; C497118:LigA protein, E coli