Site-directed mutagenesis of conserved aspartates, glutamates and arginines in the active site region of Escherichia coli DNA topoisomerase I.
To catalyze relaxation of supercoiled DNA, DNA topoisomerases form a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl group on the DNA phosphodiester backbone bond during the step of DNA cleavage. Strand passage then takes place to change the linking number. This is followed by DNA religation during which the displaced DNA hydroxyl group attacks the phosphotyrosine linkage to reform the DNA phosphodiester bond. Mg(II) is required for the relaxation activity of type IA and type II DNA topoisomerases. A number of conserved amino acids with acidic and basic side chains are present near Tyr-319 in the active site of the crystal structure of the 67-kDa N-terminal fragment of Escherichia coli DNA topoisomerase I. Their roles in enzyme catalysis were investigated by site-directed mutation to alanine. Mutation of Arg-136 abolished all the enzyme relaxation activity even though DNA cleavage activity was retained. The Glu-9, Asp-111, Asp-113, Glu-115, and Arg-321 mutants had partial loss of relaxation activity in vitro. All the mutants failed to complement chromosomal topA mutation in E. coli AS17 at 42 degreesC, possibly accounting for the conservation of these residues in evolution.
Zhu
C X
CX
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
Roche
C J
CJ
Papanicolaou
N
N
DiPietrantonio
A
A
Tse-Dinh
Y C
YC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D001224:Aspartic Acid; D001665:Binding Sites; D017931:DNA Primers; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D018698:Glutamic Acid; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D001120:Arginine; D004264:DNA Topoisomerases, Type I