title: p-Thiophenylalanine-induced DNA cleavage and religation activity of a modified vaccinia topoisomerase IB.
abstract: Vaccinia DNA topoisomerase IB is the smallest of the type IB topoisomerases. Because of its small size (314 amino acids) and target site specificity (5'(C/T)CCTTp(↓) sites), it constitutes an excellent model for studying the interaction of type IB enzymes with duplex DNA. In this study, p-thiophenylalanine was incorporated into the enzyme active site (position 274) by in vitro translation in the presence of a chemically misacylated tRNA. The modification, which resulted in replacement of the nucleophilic tyrosine OH group with SH, retained DNA topoisomerase activity and did not alter the DNA cleavage site. However, the modified topoisomerase effected relaxation of supercoiled plasmid DNA at a rate about 16-fold slower than the wild-type enzyme. The thiophenylalanine-induced DNA cleavage rate (k(cl) = 1 × 10(-4) s(-1)) was 30 times lower than for the wild-type enzyme (k(cl) = 3 × 10(-3) s(-1)). In contrast, thiophenylalanine-induced DNA religation was faster than that of the wild-type enzyme. We propose that the change in kinetics reflects the difference in bond energies between the O-P and S-P bonds being formed and broken in the reactions catalyzed by the wild-type and modified enzymes. We also studied the effect of adding Mg(2+) and Mn(2+) to the wild-type and modified topoisomerases I. Divalent metal ions such as Mg(2+) and Mn(2+) increased DNA relaxation activity of the wild-type and modified enzymes. However, the pattern of increases failed to support the possibility that metal ion-heteroatom interaction is required for catalysis.
authors.affiliation: Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States.
authors.forename: Shengxi
authors.initials: S
authors.lastname: Chen
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Sidney M
authors.initials: SM
authors.lastname: Hecht
mesh_terms: D053837:DNA Cleavage; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D009154:Mutation; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D004278:DNA, Superhelical; D013438:Sulfhydryl Compounds; D008345:Manganese; D010649:Phenylalanine; D012343:RNA, Transfer; D004264:DNA Topoisomerases, Type I; D008274:Magnesium