Covalent bonds between protein and DNA. Formation of phosphotyrosine linkage between certain DNA topoisomerases and DNA.

The cleavage of a DNA phosphodiester bond by Escherichia coli DNA topoisomerase I and the simultaneous covalent linkage of the enzyme to the 5’-phosphoryl group of the DNA at the cleavage site have been reported previously (Depew, R. E., Liu, L. F., and Wang, J. C. (1978) J. Biol. Chem 253, 511-518; Liu, L. F., and Wang, J. C. (1979) J. Biol. Chem 254, 11082-11088). With either E. coli or Micmcoccus luteus DNA topoisomerase I, formation of the covalent complex with 32P-labeled single-stranded DNA, followed by nucleolytic treatment of the complex, yields a 32P-labeled protein. The stability of the linkage between the protein and the labeled phosphorus, in aqueous buffers ranging from 1 to 13 in pH and in 3.8 M hydroxyamine at pH 4.8, suggests that the linkage is unlikely to be phosphoserine, phosphothreonine, or a phosphorus-nitrogen bond. Analysis of a 5.6 M HC1 hydrolysate of the labeled protein by paper electrophoresis and thin layer chromatography identifies 0‘“phosphotyrosine as the labeled amino acid. Thus the protein-DNA covalent bond that can form between DNA and E. coli or M. luteus DNA topoisomerase I is most likely a phosphotyrosine linkage. The sites of cleavage by the topoisomerases in a number of single-stranded DNA restriction fragments have been determined at the nucleotide sequence level. There is no nucleotide specificity on either the 3’or the 5’-side of the site of cleavage. The distribution of the cleavage sites is, however, nonrandom, and there appears to be considerable coincidence of cleavage sites for the two topoisomerases. The protein-DNA linkage formed upon cleavage of double-stranded DNA by M. luteus DNA gyrase has also been examined. It is found that the cleavage of the DNA by gyrase is accompanied by the covalent linking of subunit A, but not subunit B, of gyrase to the 5’-side of the DNA; the linkage is again a phosphotyrosine bond.