Peptide sequencing and site-directed mutagenesis identify tyrosine-727 as the active site tyrosine of Saccharomyces cerevisiae DNA topoisomerase I

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 86; no. 10; pp. 3559 - 3563
• Main Authors: Lynn, R.M, Bjornsti, M.A, Caron, P.R, Wang, J.C
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.05.1989; National Acad Sciences
• Subjects: ACIDE AMINE; Active sites; ADN; Amino Acid Sequence; AMINO ACIDS; AMINOACIDOS; Analytical, structural and metabolic biochemistry; Binding Sites; Biochemistry; Biological and medical sciences; CODE GENETIQUE; CODIGO GENETICO; DNA; DNA Mutational Analysis; DNA Topoisomerases, Type I - genetics; DNA Topoisomerases, Type I - metabolism; DNA Topoisomerases, Type I - ultrastructure; DNA, Superhelical - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - ultrastructure; ENZIMAS; ENZYME; ENZYMES; Fundamental and applied biological sciences. Psychology; GENE; GENES; Genes, Fungal; GENETIC CODE; Glycoproteins; LEVADURA; LEVURE; Molecular Sequence Data; MUTACION; MUTATION; NUCLEOTIDE; NUCLEOTIDES; NUCLEOTIDOS; PEPTIDE; PEPTIDES; PEPTIDOS; Proteins; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Sequencing; Site directed mutagenesis; TIROSINA; TYROSINE; Vaccinia; Vaccinia virus; YEASTS; Tyrosine; Chordopoxvirinae; Nucleotide sequence; Gel electrophoresis; Enzyme; Orthopoxvirus; Active site; Sequence alignment; DNA topoisomerase; Site directed mutagenesis; Fungi; Virus; Plasmid; Vaccinia virus; Enzymatic activity; Gene; Poxviridae; Fluorescent labelling; Ascomycetes; Oligonucleotide; Saccharomyces cerevisiae; Comparative study; Aminoacid sequence; Thallophyta
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.10.3559

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.