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

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.