Effect of magnesium ions and temperature on the sequence-dependent curvature of DNA restriction fragments

Abstract

Transient electric birefringence has been used to quantify the curvature of two DNA restriction fragments, a 199-base-pair fragment taken from the origin of replication of the M13 bacteriophage and a 207-base-pair fragment taken from the VP1 gene in the SV40 minichromosome. Stable curvature in the SV40 and M13 restriction fragments is due to a series of closely spaced A tracts, runs of 4-6 contiguous adenine residues located within 40 or 60 base pair 'curvature modules' near the center of each fragment. The M13 and SV40 restriction fragments exhibit bends of ∼ 45° in solutions containing monovalent cations and ∼ 60° in solutions containing Mg(2 +) ions. The curvature is not localized at a single site but is distributed over the various A tracts in the curvature modules. Thermal denaturation studies indicate that the curvature in the M13 and SV40 restriction fragments remains constant up to 30 °C in solutions containing monovalent cations, and up to 40 °C in solutions containing Mg(2 +) ions, before beginning to decrease slowly with increasing temperature. Hence, stable curvature in these DNA restriction fragments exists at the biologically important temperature of 37 °C.

Figure 1

Semilogarithmic plots of the dependence of the fractional decay of the birefringence, Δn/n, of the 199-bp SV40 fragment on the time elapsed after removal of the electric field, in μs. A, Decay observed in buffer B, without Mg²⁺ ions, τ = 3.74 μs; B, decay observed in buffer F, containing 0.1 mM Mg²⁺ ions, τ = 2.83 μs.

Figure 2

Overlapping M13 restriction fragments. A, Schematic diagram of the sequences of the overlapping 224-, 219- and 207-bp fragments. The square boxes represent A-tracts in each fragment containing 6 or more contiguous A or T residues. The apparent bend center is indicated by a vertical dashed line. B, Theoretical curves (solid lines) relating τ-ratios to bend angles and the relative distance, s, of the bend from the end of the fragment. (●), Average τ-ratios of the 224-, 219- and 207-bp M13 fragments (from top to bottom) in buffers B and C (without Mg²⁺ ions); (▲), τ-ratios of the same fragments in buffer F, with Mg²⁺ ions.

Figure 3

Overlapping SV40 restriction fragments. A, Schematic diagram of the sequences of the 199-, 204- and 206-bp fragments. The square boxes represent A-tracts in each fragment containing 6 or more A or T residues. The apparent bend center is indicated by a vertical dashed line. B, Theoretical curves (solid lines) relating τ-ratios to bend angles and the relative distance, s, of the bend from the end of the fragment. (●), Average τ-ratios of the 204-, 206- and 199-bp fragments (from top to bottom) in buffers B and C, without Mg²⁺ ions; (▲), τ-ratios of the same fragments in buffer F, with Mg²⁺ ions.

Figure 4

Dependence of the τ-ratios observed for SV40 sequence variants containing different numbers of A-tracts in the curvature module on the number of A-tracts. The error bars represent the average τ-ratios observed for sequence variants containing the same number of A-tracts. (○), τ-ratios observed in buffers B and C, without Mg²⁺ ions; and (△), τ-ratios observed in buffer F, with Mg²⁺ ions. The lines were drawn by linear regression (r² = 0.88 for buffers B and C; r² = 0.98 for buffer F).

Figure 5

Dependence of the apparent bend angles observed for curved and normal DNAs on temperature. A, buffer B, without Mg²⁺ ions; and B, buffer F, with Mg²⁺ ions. The DNAs are: (○), normal 198-bp fragment; (△), normal 209-bp fragment; (●), curved 199-bp SV40 fragment; (▲), curved 207-bp M13 fragment. The horizontal lines are drawn to guide the eye.

Scheme 1

Top: Litmus 28 sequence from 1128 to 1167 bp, containing the M13 origin of replication. Bottom: SV40 sequence from 1904 to 1964 bp. A- and T-tracts in each sequence containing four or more A or T residues without an intervening TA step are indicated by bold type and underlining. The locations of the apparent bend centers are indicated by double vertical lines.