Q.77 Which one of the following forms of the same DNA molecule would bind maximum ethidium bromide?
(A) Negatively supercoiled
(B) Covalently closed relaxed circle
(C) Linear
(D) Positively supercoiled
Negatively supercoiled DNA binds the maximum ethidium bromide among the given options. Ethidium bromide, a DNA intercalator, unwinds the double helix and preferentially binds to underwound structures to relieve torsional stress.
Options Explained
Negatively Supercoiled DNA
This form features underwound helical turns (negative supercoils), creating torsional stress that favors intercalator binding. Ethidium bromide inserts between base pairs, unwinding DNA by ~26° per molecule and relaxing negative supercoils first, allowing maximum binding until relaxation completes and excess induces positive supercoils.
Covalently Closed Relaxed Circle
Lacking supercoils, this circular DNA has no torsional stress to drive preferential binding. Ethidium bromide binds but induces compensatory positive supercoiling immediately, limiting total uptake compared to negatively supercoiled forms.
Linear DNA
Open ends prevent supercoiling, mimicking relaxed state without global topology constraints. Binding occurs evenly without stress relief motivation, resulting in lower affinity and capacity than supercoiled circles.
Positively Supercoiled DNA
Overwound structure opposes ethidium bromide’s unwinding effect, tightening the helix further and yielding the weakest binding affinity. Studies confirm intercalators bind least to positive supercoils.
Negatively supercoiled DNA binds maximum ethidium bromide due to its underwound structure that relieves torsional stress upon intercalation. This key concept in molecular biology explains ethidium bromide’s use in gel electrophoresis and topology studies.
Ethidium Bromide Binding Mechanism
Ethidium bromide intercalates between base pairs, unwinding the helix and reducing twist (Tw). In negatively supercoiled DNA (option A), this relaxes negative writhe (Wr), enabling high-affinity binding until ΔLk ≈ 0, then positive supercoiling limits further uptake—but initial capacity exceeds others. Relaxed circles (B) and linear DNA (C) lack this drive, while positively supercoiled DNA (D) resists unwinding.
| DNA Form | Superhelical Density (σ) | EtBr Binding Affinity | Reason |
|---|---|---|---|
| Negatively Supercoiled | Negative | Highest | Relaxes underwinding |
| Relaxed Circle | 0 | Moderate | No stress relief |
| Linear | N/A (no topology) | Low | Even binding |
| Positively Supercoiled | Positive | Lowest | Opposes unwinding |
Practical Implications in Biotechnology
In agarose gel electrophoresis with ethidium bromide, supercoiled plasmids migrate fastest due to compactness, but binding saturates negatively supercoiled forms most, aiding topology visualization. Topoisomerase assays exploit this preference.
Why Option (A) is Correct
Quantitative studies via competition dialysis show ethidium bromide partitions >10-fold more into negatively supercoiled vs positively supercoiled DNA, confirming maximum binding.
