Mutation in Cys662 ofEscherichia coliDNA Topoisomerase I Confers Temperature Sensitivity and Change in DNA Cleavage Selectivity

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Abstract

Cys662 is one of the 12 cysteine residues proposed to be co-ordination sites for binding of three Zn(II) inEscherichia coliDNA topoisomerase I. Oligonucleotide-directed mutagenesis was used to convert Cys662 to either serine or histidine. The mutant enzymes were overexpressed and purified to homogeneity. Analysis of the purified enzymes demonstrated that the mutations resulted in loss of one tightly bound Zn(II).In vivocomplementation tests andin vitrorelaxation activity assays at different temperatures showed that the partial relaxation activities retained in the two mutant enzymes were temperature sensitive. Fluorescence measurements indicated that the wild-type and mutant enzymes have structural differences. When DNA cleavage specificity was examined, the mutant enzymes were found to have altered cleavage site preferences. The preferred cleavage sites of the wild-type enzyme all had a cytosine residue four nucleotides to the minus side of the break. The cleavage sites produced by the mutant enzymes did not show a preference for cytosine at that position.f2

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Cited by (22)

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Abbreviations used: ICP, inductively coupled plasma; PAR, 4-(2-pyridylazo)resorcinol; PMPS,p-(hydroxymercuri)benzenesulfonate.

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