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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. Our new method reveals negative supercoiling increases CRISPR-Cas9 off-target effects, while both positive and negative supercoiling enhance MutS binding to mismatched DNA.

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Area of Science:

  • Molecular Biology
  • Biophysics

Background:

  • DNA supercoiling is a critical factor influencing DNA metabolism and protein interactions.
  • Understanding supercoiling's role at the single-molecule level is essential for elucidating DNA-protein dynamics.

Purpose of the Study:

  • To develop a robust protocol for site-specific DNA modification and supercoiling induction.
  • To investigate the impact of DNA supercoiling on the functional activities of CRISPR-Cas9 and MutS proteins.

Main Methods:

  • Site-specific labeling of plasmid DNA with fluorophores and biotin.
  • Induction of negative and positive supercoiling using gyrase and reverse gyrase.
  • Single-molecule analysis of protein-DNA interactions under varying supercoiling conditions.

Main Results:

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

Conclusions:

  • The developed protocol provides a versatile tool for studying supercoiling effects on DNA-protein interactions.
  • DNA supercoiling differentially modulates the activities of DNA-interacting proteins like Cas9 and MutS.
  • These findings offer new insights into the regulatory role of DNA topology in biological processes.