Monovalent cation binding by curved DNA molecules containing variable numbers of a-tracts

Abstract

Monovalent cation binding by DNA A-tracts, runs of four or more contiguous adenine or thymine residues, has been determined for two curved approximately 200 basepair (bp) restriction fragments, one taken from the M13 origin of replication and the other from the VP1 gene of SV40. These two fragments have previously been shown to contain stable, centrally located bends of 44 degrees and 46 degrees , respectively, located within approximately 60 bp "curvature modules" containing four or five irregularly spaced A-tracts. Transient electric birefringence measurements of these two fragments, sequence variants containing reduced numbers of A-tracts in the SV40 curvature module or changes in the residues flanking the A-tracts in the M13 curvature module, have been combined with the free solution electrophoretic mobilities of the same fragments using known equations to estimate the effective charge of each fragment. The effective charge is reduced, on average, by one-third charge for each A-tract in the curvature module, suggesting that each A-tract binds a monovalent cation approximately one-third of the time. Monovalent cation binding to two or more A-tracts is required to observe significant curvature of the DNA helix axis.

Figure 1

Analysis of the birefringence decay curve observed for the parent 199-bp SV40 fragment in 1 mM Tris-Cl buffer. The logarithm of the fractional birefringence signal remaining after time, t, is plotted as a function of the time after removal of the electric field. The birefringence decay curve is monoexponential, as shown by the straight line through the data points; the relaxation time is 3.74 ± 0.06 μs. The inset shows the applied 8.7 μs square-wave pulse (dashed line) and the corresponding birefringence signal (noisier curved line). Adapted from Lu et al. with permission.

Figure 2

Comparison of the free solution mobility of the parent SV40 (199-bp) and M13 (217-bp) restriction fragments (●) with the mobilities of normal, non-A-tract-containing fragments of various sizes (○), measured in 13 mM TAE buffer. The error bars represent the standard deviation of triplicate measurements; the straight line is drawn to guide the eye. Inset: Typical electropherograms observed for the curved 199-bp SV40 fragment (right peak) and oligomer 199K, which had all five A-tracts in the curvature module mutated to other sequences (left peak). The optical density, in arbitrary units, is plotted as a function of migration time.

Figure 3

Dependence of the change in the average number of bound cations, ΔN, on the number of A-tracts in the curvature module. (○) SV40 sequence variants; (▵), M13 sequence variants. The error bars correspond to the standard deviation of the ΔN values observed for sequence variants containing the same number of A-tracts. The line was drawn by linear regression (r² = 0.96); the slope of the line is 0.31.

Figure 4

The average number of cations bound by SV40 (○) and M13 (▵) sequence variants containing the same number of A-tracts is plotted as a function of (A) the average polyacrylamide gel mobility decrements, Δμ/μ, and (B) the average τ-decrements, Δτ/τ, observed for the same fragments. The error bars represent the average deviation of the ΔN, Δμ/μ, and Δτ/τ values observed for sequence variants containing the same number of A-tracts. The straight line in (A) was drawn by linear regression (r² = 0.96). The dotted line in (B) is a sigmoidal fit of the data points.

Scheme 1

Sequences of the curvature modules in the VP1 gene of SV40 and the M13 origin of replication