Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting.

Area of Science:

• Molecular Biology
• Biophysics

Background:

• Cellular DNA metabolism requires a balance between double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA).
• Traditional DNA melting studies use heat, showing AT-rich sequences melt more readily than GC-rich sequences.
• Cellular DNA melting is primarily driven by negative supercoiling, not temperature.

Purpose of the Study:

• To investigate the effect of GC content on DNA melting induced by negative supercoiling.
• To understand how DNA sequence influences melting under force and supercoiling.

Main Methods:

• Single-molecule magnetic tweezer measurements were used to study DNA molecules with varying GC content (38%–77%).
• DNA length was measured as a function of applied stretching force and supercoiling density.
• The characteristic force (Fchar) at which melting initiates was determined for different sequences.

Main Results:

• At low forces, negative supercoiling induces DNA twisting and loop formation (plectonemes) irrespective of sequence.
• Increasing force with negative supercoiling induces local DNA melting.
• Surprisingly, GC-rich sequences melted at lower forces (0.56 pN for 77% GC) than AT-rich sequences (0.73 pN for 38% GC).

Conclusions:

• The counterintuitive melting behavior is explained by denaturation bubbles forming in local AT-rich regions.
• Increased DNA bending/torsional energy in plectonemes associated with GC-rich sequences facilitates melting.
• This suggests that GC-rich regions adjacent to AT-rich regions enhance local DNA helix opening.