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Studies of the topoisomerase II-mediated cleavage and religation reactions by use of a suicidal double-stranded DNA substrate

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  • A H Andersen
  • B S Sørensen, Molekylærbiologisk Institut, Denmark
  • K Christiansen
  • ,
  • J Q Svejstrup, Molekylærbiologisk Institut, Denmark
  • K Lund, Molekylærbiologisk Institut, Denmark
  • O Westergaard

The cleavage and religation reactions of eukaryotic topoisomerase II were studied by use of a 5'-recessed DNA substrate containing a strong recognition sequence for the enzyme. Cleavage of the DNA substrate was suicidal, that is the enzyme was unable to religate the cleaved DNA due to a release of DNA 5' to the cleavage position. With this substrate cleavage products accumulated with time in the absence of protein-denaturing agents, and the cleavage reaction was not reversible with salt. The suicide cleavage complexes contained a kinetically competent topoisomerase II enzyme as determined by the enzyme's ability to perform intermolecular ligation of the cleaved DNA to a free 3'-hydroxyl end on another DNA strand. The efficiency of the religation reaction depended on the ability of the religation substrate to base pair to the DNA in the cleaved enzyme-DNA complex. Higher levels of religation were obtained with dinucleotides than with long DNA substrates. Mononucleotides also were efficiently religated, indicating an ability of the enzyme to mediate religation without making contacts to a long stretch of nucleotides 5' to the cleavage position.

Original languageEnglish
JournalJournal of Biological Chemistry
Volume266
Issue14
Pages (from-to)9203-10
Number of pages8
ISSN0021-9258
Publication statusPublished - 15 May 1991

    Research areas

  • Animals, Base Sequence, Cattle, DNA, DNA Topoisomerases, Type II, In Vitro Techniques, Molecular Sequence Data, Sodium Chloride, Sodium Dodecyl Sulfate, Structure-Activity Relationship, Thymus Gland, Time Factors, Journal Article, Research Support, Non-U.S. Gov't

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