Abstract
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.
Highlights
Important insights into the potential enzyme-phosphate contacts have come from several crystal structures of E. coli topoisomerase I and III
Previous site-directed mutagenesis experiments from different laboratories have characterized a number of conserved residues in E. coli DNA topoisomerase I that were proposed to be interacting with the scissile phosphate in the DNA cleavage mechanism [23,24,25]
The positively charged lysine substituted at position 195 might be able to participate in an electrostatic interaction with DNA, but it could not position the G strand DNA correctly for DNA cleavage to take place, perhaps because of the change in enzyme folding caused by the arginine to lysine substitution, as indicated by the proteolytic digestion data
Summary
Important insights into the potential enzyme-phosphate contacts have come from several crystal structures of E. coli topoisomerase I and III. 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
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