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Related Experiment Videos

Hin-mediated inversion on positively supercoiled DNA

H M Lim1, H J Lee, C Jaxel

  • 1Department of Biology, College of Natural Sciences, Chungnam National University, 305-764 Taejon, Republic of Korea. hmlim@hanbat.chungnam.ac.kr

The Journal of Biological Chemistry
|July 18, 1997
PubMed
Summary
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Hin recombinase efficiently inverts DNA, but requires negatively supercoiled DNA. Positively supercoiled DNA significantly hinders the strand exchange step, suggesting a need for DNA unwinding post-cleavage for efficient inversion.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • DNA supercoiling is crucial for various DNA metabolic processes.
  • Hin recombinase mediates DNA inversion, a process essential for gene regulation in some organisms.
  • The superhelical state of DNA can influence the efficiency of DNA-protein interactions and enzymatic activities.

Purpose of the Study:

  • To investigate the role of DNA supercoiling, specifically positive supercoiling, on Hin-mediated DNA inversion.
  • To determine how positive supercoiling affects the DNA binding, cleavage, and strand exchange activities of Hin recombinase.
  • To elucidate the mechanistic requirements for Hin recombinase activity in relation to DNA topology.

Main Methods:

  • Generation of positively supercoiled plasmid DNA using reverse gyrase from Sulfolobus shibatae.

Related Experiment Videos

  • In vitro Hin-mediated DNA inversion assays using both negatively and positively supercoiled substrates.
  • Analysis of Hin and Fis DNA binding activity.
  • Stepwise analysis of the inversion reaction, including DNA cleavage and strand exchange.

    • Main Results:

    • Hin and Fis proteins exhibited similar DNA binding affinities to both negatively and positively supercoiled plasmids.
    • DNA inversion activity on positively supercoiled DNA was less than 1% compared to negatively supercoiled DNA.
    • Hin recombinase efficiently cleaved recombination sites on positively supercoiled DNA but failed to perform strand exchange.

    Conclusions:

    • Positive DNA supercoiling significantly inhibits the strand exchange step of Hin-mediated inversion.
    • The data suggest that DNA unwinding at the recombination site after cleavage is a critical step for strand exchange to occur.
    • This finding highlights the importance of DNA topology in regulating the function of site-specific recombinases like Hin.