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A high-throughput single-molecule platform to study DNA supercoiling effect on protein-DNA interactions.

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DNA supercoiling impacts protein interactions. Negative supercoiling increases CRISPR-Cas9 off-target effects, while both negative and positive supercoiling enhance MutS binding to mismatched DNA.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • DNA supercoiling is a critical factor in DNA metabolism and protein interactions.
  • Understanding supercoiling's influence on DNA-protein dynamics is essential for various biological processes.

Purpose of the Study:

  • To investigate the effects of DNA supercoiling on the function of CRISPR-Cas9 and the mismatch repair protein MutS at the single-molecule level.
  • To develop and validate a novel protocol for site-specific DNA modification and supercoiling induction.

Main Methods:

  • Developed a protocol for site-specific labeling of plasmid DNA with fluorophores and biotin.
  • Induced physiological levels of negative and positive supercoiling using gyrase and reverse gyrase.
  • Assessed DNA-protein interactions using single-molecule techniques, comparing supercoiled DNA with relaxed DNA.

Main Results:

  • Negative DNA supercoiling significantly increased off-target DNA unwinding by CRISPR-Cas9.
  • Both negative and positive supercoiling enhanced the binding of MutS to mismatched base pairs.
  • Supercoiling did not alter the rate of ATP-induced sliding clamp formation for MutS.

Conclusions:

  • The developed protocol is effective for studying supercoiling effects on DNA-protein interactions.
  • DNA supercoiling plays a crucial role in modulating the specificity and function of DNA-interacting proteins like Cas9 and MutS.
  • Findings provide new insights into the dynamics of protein-DNA interactions under varying supercoiling conditions.