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Topoisomerases are enzymes that relax overwound DNA molecules during various cell processes, including DNA replication and transcription. These enzymes regulate positive and negative DNA supercoiling without changing the nucleotide sequence. DNA overwinding in a clockwise direction results in positively supercoiled DNA, whereas underwinding in a counterclockwise direction produces negatively supercoiled DNA.
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DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
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Topoisomerase II N-terminal ATPase Clamp Stabilization.

Lauren A Fielding1, Joseph Deweese2,3

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|May 15, 2025
PubMed
Summary

This study introduces a topoisomerase II assay to measure enzyme-DNA complex stability. The method quantifies how different conditions affect the strength of the topoisomerase-DNA interaction.

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ATPaseDNA bindingInhibitionPlasmid DNATopoisomerase II

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Topoisomerase II enzymes are crucial for DNA replication and repair.
  • Understanding the stability of topoisomerase-DNA interactions is key to comprehending enzyme function.

Purpose of the Study:

  • To develop and validate a novel assay for assessing topoisomerase II-DNA complex stability.
  • To investigate factors influencing the stabilization of the N-terminal ATPase clamp interaction with DNA.

Main Methods:

  • A topoisomerase II N-terminal ATPase clamp stabilization assay was employed.
  • Protein:DNA complexes were immobilized and subjected to washes of increasing stringency.
  • DNA eluted at each wash step was quantified via gel electrophoresis.

Main Results:

  • The assay effectively determined the relative stability of topoisomerase II-DNA interactions.
  • The stringency of washes correlated with the disruption of enzyme:DNA complexes.
  • The method allows for the identification of conditions that stabilize these complexes.

Conclusions:

  • The developed assay provides a robust method for studying enzyme-DNA complex dynamics.
  • This technique facilitates the exploration of regulatory mechanisms governing topoisomerase II activity.
  • The findings contribute to a deeper understanding of DNA topology management by topoisomerase II.