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In vitro evolution of preferred topoisomerase II DNA cleavage sites
1Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Abstract:
Topoisomerase II is an essential enzyme that is the target for several clinically important anticancer drugs. Although this enzyme must create transient double-stranded breaks in the genetic material in order to carry out its indispensable DNA strand passage reaction, the factors that underlie its nucleotide cleavage specificity remain an enigma. Therefore, to address the critical issue of enzyme specificity, a modified systematic evolution of ligands by exponential enrichment (SELEX) protocol was employed to select/evolve DNA sequences that were preferentially cleaved by Drosophila melanogaster topoisomerase II. Levels of DNA scission rose substantially (from 3 to 20%) over 20 rounds of SELEX. In vitro selection/evolution converged on an alternating purine/pyrmidine sequence that was highly AT-rich (TATATATACATATATATA). The preference for this sequence was more pronounced for Drosophila topoisomerase II over other species and was increased in the presence of DNA cleavage-enhancing anticancer drugs. Enhanced cleavage appeared to be based on higher rates of DNA scission rather than increased binding affinity or decreased religation rates. The preferred sequence for topoisomerase II-mediated DNA cleavage is dramatically overrepresented ( approximately 10,000-fold) in the euchromatic genome of D. melanogaster, implying that it may be a site for the physiological action of this enzyme.
Insights
Researchers identified specific DNA sequences preferentially cleaved by Drosophila melanogaster topoisomerase II using modified SELEX. This AT-rich sequence is overrepresented in the genome, suggesting a role in the enzyme's physiological function.
Area of Science:
- Molecular Biology
- Genomics
- Enzymology
Background:
- Topoisomerase II is a crucial enzyme and a target for anticancer drugs.
- The enzyme's nucleotide cleavage specificity is not well understood.
- Understanding specificity is key to developing targeted therapies.
Purpose of the Study:
- To elucidate the nucleotide sequence specificity of Drosophila melanogaster topoisomerase II.
- To identify DNA sequences preferentially cleaved by this enzyme.
- To investigate the impact of anticancer drugs on enzyme specificity.
Main Methods:
- Modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was used to evolve DNA sequences.
- Selection was performed over 20 rounds to enrich for cleavage-preferred sequences.
- DNA cleavage assays were conducted with and without anticancer drugs.
Main Results:
- SELEX enriched for a highly AT-rich, alternating purine/pyrimidine sequence (TATATATACATATATATA).
- This sequence preference was specific to Drosophila topoisomerase II and enhanced by anticancer drugs.
- Increased cleavage was attributed to higher scission rates, not altered binding or religation.
Conclusions:
- A specific AT-rich DNA sequence is preferentially cleaved by Drosophila topoisomerase II.
- This sequence is significantly overrepresented in the D. melanogaster euchromatic genome.
- The findings suggest a potential physiological role for this sequence in topoisomerase II activity.