Site-specific interaction of vaccinia virus topoisomerase I with duplex DNA. Minimal DNA substrate for strand cleavage in vitro.
Purified vaccinia virus DNA topoisomerase I forms a cleavable complex with duplex DNA at a conserved sequence element 5'(C/T)CCTTdecreases in the incised DNA strand. DNase I footprint studies show that vaccinia topoisomerase protects the region around the site of covalent adduct formation from nuclease digestion. On the cleaved DNA strand, the protected region extends from +13 to -13 (+1 being the site of cleavage). On the noncleaved strand, the protected region extends from +13 to -9. Similar nuclease protection is observed for a mutant topoisomerase (containing a Tyr ---- Phe substitution at the active site amino acid 274) that is catalytically inert and does not form the covalent intermediate. Thus, vaccinia topoisomerase is a specific DNA binding protein independent of its competence in transesterification. By studying the cleavage of a series of 12-mer DNA duplexes in which the position of the CCCTTdecreases motif within the substrate is systematically phased, the "minimal" substrate for cleavage has been defined; cleavage requires six nucleotides upstream of the cleavage site and two nucleotides downstream of the site. An analysis of the cleavage of oligomer substrates mutated singly in the CCCTT sequence reveals a hierarchy of mutational effects based on position within the pentamer motif and the nature of the sequence alteration.
Shuman
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Program in Molecular Biology, Sloan-Kettering Institute, New York, New York 10021.
D001483:Base Sequence; D001665:Binding Sites; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000201:enzymology*
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004264:DNA Topoisomerases, Type I