Site-directed mutagenesis of residues involved in G Strand DNA binding by Escherichia coli DNA topoisomerase I.
Crystal structures of complexes between type IA DNA topoisomerases and single-stranded DNA suggest that the residues Ser-192, Arg-195, and Gln-197 in a conserved region of Escherichia coli topoisomerase I may be important for direct interactions with phosphates on the G strand of DNA, which is the substrate for DNA cleavage and religation (Changela A., DiGate, R. J., and Mondragón, A. (2001) Nature 411, 1077-1081; Perry, K., and Mondragón, A. (2003) Structure 11, 1349-1358). Site-directed mutagenesis experiments altering these residues to alanines and other amino acids were carried out to probe the relevance of these interactions in the catalytic activities of the enzyme. The results show that the side chains of Arg-195 and Gln-197 are required for DNA cleavage by the enzyme and are likely to be important for positioning of the G strand of DNA at the active site prior to DNA cleavage. Mutation of Ser-192 did not affect DNA binding and cleavage but nevertheless decreased the overall rate of relaxation of supercoiled DNA probably because of its participation in a later step of the reaction pathway.
Cheng
Bokun
B
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
Feng
Jingyang
J
Mulay
Vishwaroop
V
Gadgil
Sharvari
S
Tse-Dinh
Yuk-Ching
YC
D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics; D001665:Binding Sites; D003461:Crystallography; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates / Q000494:pharmacology; D005973:Glutamine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity
D005971:Glutamates; D005973:Glutamine; D012694:Serine; D004247:DNA; D001120:Arginine; D004264:DNA Topoisomerases, Type I