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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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For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
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DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...
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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
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Profiling DNA supercoiling domains in vivo.

Samuel Corless1, Catherine Naughton1, Nick Gilbert1

  • 1Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.

Genomics Data
|October 21, 2015
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Summary

Researchers developed a new method to map DNA supercoiling, a key factor in gene regulation. This technique reveals the large-scale distribution of DNA supercoiling in human cells for the first time.

Keywords:
DNADomainsHumanPull-downSupercoiling

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Area of Science:

  • Molecular Biology
  • Genetics
  • Epigenetics

Background:

  • DNA structure transitions, such as supercoiling, are crucial for gene regulation but remain poorly understood in eukaryotes.
  • DNA supercoiling, the winding of the DNA double helix, directly impacts transcription initiation, elongation, and gene expression.
  • This process is influenced by polymerase activity and modulated by topoisomerase enzymes.

Purpose of the Study:

  • To develop and detail a novel technique for mapping the distribution of DNA supercoiling within cell nuclei.
  • To characterize the large-scale distribution of DNA supercoiling in human cells using this new method.

Main Methods:

  • Development of biotinylated 4,5,8-trimethylpsoralen (bTMP) pull-down assay.
  • The bTMP method is designed to selectively enrich for under-wound DNA regions.
  • Detailed description of experimental design, quality control measures, and data analysis procedures.

Main Results:

  • The study successfully characterized the large-scale distribution of DNA supercoiling in human cells.
  • Provided a detailed account of the methodology enabling this characterization.

Conclusions:

  • The developed bTMP pull-down technique offers a powerful new tool for investigating DNA supercoiling.
  • This work provides the first comprehensive map of DNA supercoiling distribution in human cells, opening avenues for understanding its regulatory roles.