Modification of nucleic acid structure by DNA topoisomerase l

Krislina A. Henninqteld, Tuncer Asian, Sidney M. Hecht

Research output: Contribution to journalArticle

Abstract

DNA topoisomerases modulate the topological structure of DNA through transient breaks in the phosphodiester backbone of DNA. Mechanistically, the single-strand breaks mediated by eukaryotic DNA topoisomerases I involve reversible formation of an intermediate in which the active site tyrosine is linked to DNA covalently via a 3'-O-phosphotyrosine bond. The ligation reaction of topoisomerase I was characterized utilizing partial duplexes which uncouple the cleavage and ligation reactions of the enzyme, thereby trapping the intermediate. Reversal of DNA cleavage results from the addition of acceptor oligomers containing a free 5'-OH and capable of hybridizing to the noncleaved strand of the 'suicide substrate". Modification of nucleic acid structure by DNA topoisomerase I has resulted from the use of partially complementary acceptor oligomers. These transformations include the formation of DNA insertions, deletions and mismatches, and constitute a reasonable model system for topoisomerase I - mediated illegitimate recombination. To further define the capacity of topoisomerase I to catalyze structural transformations of DNA, nucleophiles other than 5'-OH were studied as substrates for the topoisomerase I reverse reaction. Toward this end, the topoisomerase I - DNA covalent intermediate was purified to homogeneity, allowing investigation of the ligation reaction independent of the forward reaction. In addition, oligonucleotides containing 5'-thiol, -amino and -homo moieties were synthesized. All of the modified acceptors facilitated displacement of the enzyme - linked tyrosine moiety with concomitant alteration in the internucleosidic bond. Verification of the topoisomerase I mediated modified linkages were obtained by both chemical and enzymatic degradation analysis.

Original languageEnglish (US)
Pages (from-to)A1241
JournalFASEB Journal
Volume10
Issue number6
StatePublished - Dec 1 1996
Externally publishedYes

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ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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