Mapping the active site tyrosine of Escherichia coli DNA gyrase.

We have identified tyrosine 122 of the A subunit of Escherichia coli DNA gyrase as the tyrosine that becomes covalently bound to DNA when the enzyme breaks the phosphodiester bonds of DNA. The covalent gyrase X DNA complex was isolated following cleavage of the DNA by gyrase in the presence of the gyrase inhibitor oxolinic acid. The active site tyrosine was first mapped to two overlapping peptides. Its precise position in the sequence of the A subunit of gyrase was then determined by sequencing of a peptide bound to DNA. We also present a method for mapping sites of DNA attachment in a protein of known amino acid sequence. The covalent complex of DNA and protein is treated with proteases that cut specifically. The electrophoretic mobilities of the resulting peptide-bound DNA molecules are correlated with the sizes of the bound peptides, allowing determination of the site of attachment of the DNA.