Intermolecular DNA ligation activity of eukaryotic toposiomerase II: Potential roles in nucleic acid recombination

Miscellaneous · Wed Jan 01 00:00:00 EST 1992

OSTI ID:5507453

Gale, K C.R.

Single-stranded [phi]X174 (+) strand DNA was used as a model substrate for topoisomerase II to determine whether double-stranded DNA cleavage observed in vitro reflects the in vivo intermediate in the enzyme's catalytic cycle and to investigate potential mechanisms for topoisomerase II-mediated DNA recombination. As found previously for topoisomerase II-mediated cleavage of double-stranded DNA, the enzyme was covalently linked to the 5[prime]-termini of cleaved [phi]X174 molecules. Optimal reaction conditions were similar for the two substrates. In contrast to results with double-stranded molecules, single-stranded DNA cleavage increased with time, was not reversible, and did not require the presence of SDS. Cleavage products generated in the absence of protein denaturant contained free 3[prime]-OH DNA termini. These results strongly suggest that the covalent topoisomerase II-cleaved DNA complex observed in vitro is the active intermediate in the enzyme's catalytic code. Topoisomerase II is capable of joining cleaved [phi]X174 (+) strand DNA to duplex oligonucleotide acceptor molecules by an intermolecular ligation reaction. Intermolecular DNA ligation proceeded in a time and oligonucleotide concentration dependent fashion. The covalent linkage is between the 5[prime]-phosphate of [phi]X174 (+) strand DNA and the 3[prime]-OH of oligonucleotide acceptor molecules. The reaction was dependent on the presence of a divalent cation, was inhibited by salt, and was not affected by the presence of ATP. The enzyme was capable of ligating [phi]X174 (+) strand DNA to double-stranded oligonucleotides that contained 5[prime]-overhang, 3[prime]-overhang, or blunt ends. Single-stranded, nicked, or gapped oligonucleotides could also be used as acceptor molecules. These results demonstrate that the type II enzyme has an intrinsic ability to mediate illegitimate DNA recombination in vitro and suggests possible roles for topoisomerase II in nucleic acid recombination in vivo.

• Research Organization: Vanderbilt Univ., Nashville, TN (United States)
• OSTI ID: 5507453
• Resource Relation: Other Information: Thesis (Ph.D.)
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
37 INORGANIC
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ISOMERASES
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GENE RECOMBINATION
BIOCHEMICAL REACTION KINETICS
BIOCHEMISTRY
DNA
IN VITRO
IN VIVO
LIGASES
OLIGONUCLEOTIDES
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