Peptide sequencing and site-directed mutagenesis identify tyrosine-727 as the active site tyrosine of Saccharomyces cerevisiae DNA topoisomerase I.
Extensive digestion of the covalent intermediate between DNA and Saccharomyces cerevisiae DNA topoisomerase I with trypsin yields a 7-amino acid peptide covalently linked to DNA. Direct sequencing of the DNA-linked peptide identifies Tyr-727 as the active site tyrosine that forms an O4-phosphotyrosine bond with DNA when the enzyme cleaves a DNA phosphodiester bond. Site-directed mutagenesis of the cloned yeast TOP1 gene encoding the enzyme confirms the essentiality of Tyr-727 for the relaxation of supercoiled DNA by the enzyme. From amino acid sequence homology, Tyr-771 and -773 are readily identified as the active site tyrosines of Schizosaccharomyces pombe and human DNA topoisomerase I, respectively. Sequence comparison and site-directed mutagenesis also implicate Tyr-274 of vaccinia virus DNA topoisomerase as the active site residue. There appears to be a 70-amino acid domain near the carboxyl terminus of eukaryotic DNA topoisomerase I and vaccinia topoisomerase, within which the active site tyrosine resides.
Lynn
R M
RM
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Bjornsti
M A
MA
Caron
P R
PR
Wang
J C
JC
D000595:Amino Acid Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000648:ultrastructure; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014443:Tyrosine
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D014443:Tyrosine; D004264:DNA Topoisomerases, Type I