MUTATION IN CYS662 OF ESCHERICHIA-COLI DNA TOPOISOMERASE-I CONFERS TEMPERATURE SENSITIVITY AND CHANGE IN DNA CLEAVAGE SELECTIVITY

Cys662 is one of the 12 cysteine residues proposed to be co-ordination sites for binding of three Zn(II) in Escherichia coli DNA topoisomera se I. Oligonucleotide-directed mutagenesis was used to convert Cys662 to either serine or histidine. The mutant enzymes were overexpressed a nd purified to homogeneity. Analysis of the purified enzymes demonstra ted that the mutations resulted in loss of one tightly bound Zn(lI). I n vivo complementation tests and in vitro relaxation activity assays a t different temperatures showed that the partial relaxation activities retained in the two mutant enzymes were temperature sensitive. Fluore scence measurements indicated that the wild-type and mutant enzymes ha ve structural differences. When DNA cleavage specificity was examined, the mutant enzymes were found to have altered cleavage site preferenc es. The preferred cleavage sites of the wild-type enzyme all had a cyt osine residue four nucleotides to the minus side of the break. The cle avage sites produced by the mutant enzymes did not show a preference f or cytosine at that position. (C) 1995 Academic Press Limited