Peptide sequencing and site-directed mutagenesis identify tyrosine-727 as the active site tyrosine of Saccharomyces cerevisiae DNA topoisomerase I.
AUTOR(ES)
Lynn, R M
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=287177Documentos Relacionados
- Site-Directed Mutagenesis of a Saccharomyces Cerevisiae Mitochondrial Translation Initiation Codon
- In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis.
- Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I.
- Site-directed mutagenesis of polyomavirus middle-T antigen sequences encoding tyrosine 315 and tyrosine 250.
- The use of native T7 DNA polymerase for site-directed mutagenesis.
