Ligation of synthetic activated DNA substrates by site-specific recombinases and topoisomerase I.
The FLP protein of the 2-microns plasmid of Saccharomyces cerevisiae is a conservative site-specific recombinase that is involved in the amplification of the plasmid. This recombination reaction proceeds via the covalent attachment of the protein to the 3'-phosphoryl group at the site of the breaks through a phosphotyrosine linkage. We have recently developed an assay that measures FLP-mediated strand ligation independent of FLP-mediated cleavage and covalent attachment to the DNA. The substrate for ligation was produced by FLP-induced cleavage of the FLP recognition site followed by digestion with Pronase and was shown to contain (at least) a tyrosine residue at the 3'-PO4 terminus adjacent to the FLP cleavage sites. We have now synthesized artificial substrates that bear a tyrosine residue on the 3'-PO4 of an appropriate oligonucleotide and find that this substrate is ligated as efficiently as the previous ligation substrates that were isolated after FLP cleavage of the substrate. Analogous substrates for other members of the integrase family of recombinases (lambda integrase protein, P1-Cre protein) as well as for mammalian topoisomerase I are also active as ligation substrates with their cognate protein. This class of activated substrates should be useful in the study of breakage and reunion reactions involving DNA.
Department of Molecular and Medical Genetics, University of Toronto, Canada.
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D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D007202:Indicators and Reagents; D019426:Integrases; D008025:Ligases / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D045522:Recombinases; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology
D005656:Fungal Proteins; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D045522:Recombinases; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases; C115006:integron integrase IntI1; D004264:DNA Topoisomerases, Type I; D008025:Ligases