Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins

Area of Science:

• Molecular Biology
• Biophysics
• Genetics

Background:

• DNA supercoiling influences protein activity by locally unwinding the DNA double helix.
• Previous studies lacked nucleotide-level resolution of DNA deformation and unpaired bases induced by supercoiling.

Purpose of the Study:

• To locate unpaired bases in DNA mini-circles (dsMCs) across varying supercoiling states.
• To characterize the binding of DNA metabolism proteins to supercoiled DNA.

Main Methods:

• Utilized radiolabeled double-stranded DNA mini-circles (dsMCs) to analyze topology.
• Investigated protein binding with and without magnesium to assess divalent cation effects.
• Employed Nuclease SI and protein binding assays (E. coli topoisomerase I, human replication protein A (hRPA), E. coli SSB).

Main Results:

• Nuclease SI activity increased nearly tenfold on hyper-negatively supercoiled DNA compared to relaxed DNA.
• Structural changes were mapped at base-pair resolution, showing subtle, sequence-distributed alterations.
• Escherichia coli topoisomerase I binding increased with negative supercoiling, while human replication protein A (hRPA) binding showed a strong positive correlation with negative supercoiling.
• Magnesium presence altered DNA topology and significantly modified hRPA and E. coli SSB binding, indicating magnesium-dependent interactions.

Conclusions:

• DNA supercoiling induces subtle, distributed structural changes at the nucleotide level, influencing enzyme activity.
• Protein binding to DNA is modulated by DNA topology (supercoiling) and the presence of divalent cations like magnesium.
• Human replication protein A (hRPA) binding is twist-dependent, and its association with DNA does not alter the DNA's overall topology.