Intrinsic curvature in the VP1 gene of SV40: comparison of solution and gel results

Abstract

DNA restriction fragments that are stably curved are usually identified by polyacrylamide gel electrophoresis because curved fragments migrate more slowly than normal fragments containing the same number of basepairs. In free solution, curved DNA molecules can be identified by transient electric birefringence (TEB) because they exhibit rotational relaxation times that are faster than those of normal fragments of the same size. In this article, the results observed in free solution and in polyacrylamide gels are compared for a highly curved 199-basepair (bp) restriction fragment taken from the VP1 gene in Simian Virus 40 (SV40) and various sequence mutants and insertion derivatives. The TEB method of overlapping fragments was used to show that the 199-bp fragment has an apparent bend angle of 46 +/- 2 degrees centered at sequence position 1922 +/- 2 bp. Four unphased A- and T-tracts and a mixed A3T4-tract occur within a span of approximately 60 bp surrounding the apparent bend center; for brevity, this 60-bp sequence element is called a curvature module. Modifying any of the A- or T-tracts in the curvature module by site-directed mutagenesis decreases the curvature of the fragment; replacing all five A- and T-tracts by random-sequence DNA causes the 199-bp mutant to adopt a normal conformation, with normal electrophoretic mobilities and birefringence relaxation times. Hence, stable curvature in this region of the VP1 gene is due to the five unphased A- and T- tracts surrounding the apparent bend center. Discordant solution and gel results are observed when long inverted repeats are inserted within the curvature module. These insertion derivatives migrate anomalously slowly in polyacrylamide gels but have normal, highly flexible conformations in free solution. Discordant solution and gel results are not observed if the insert does not contain a long inverted repeat or if the long inverted repeat is added to the 199-bp fragment outside the curvature module. The results suggest that long inverted repeats can form hairpins or cruciforms when they are located within a region of the helix backbone that is intrinsically curved, leading to large mobility anomalies in polyacrylamide gels. Hairpin/cruciform formation is not observed in free solution, presumably because of rapid conformational exchange. Hence, DNA restriction fragments that migrate anomalously slowly in polyacrylamide gels are not necessarily stably curved in free solution.

FIGURE 1

Electrophoretic mobility of the permuted sequence isomers of SV40 in: (A) 1% agarose and (B) 5.7% T, 1% C polyacrylamide gels. The leftmost lanes in panel A contain the 1-kb ladder and the HinfI digest of pBR322 as mobility markers; the rightmost lane in panel B contains the 1-kb ladder. The other lanes contain SV40 linearized with BanI, HpaII, PpuMI, EcoRV, HaeII, DrdI, AccI, AflII, EcoRI, XcmI, Bsp120I, BamHI, and BclI, from left to right.

FIGURE 2

Mobilities of the permuted sequence isomers of SV40 plotted as a function of the restriction enzyme cut site, in kilobases. From left to right, the solid circles correspond to the mobilities of sequence isomers linearized by BanI, HpaII, PpuMI, EcoRV, HaeII, DrdI, AccI, AflII, EcoRI, XcmI, Bsp120I, BamHI, BclI, TaqI, and BglI, electrophoresed in a 3.5% T, 1% C polyacrylamide gel cast and run in TBE buffer at 4°C. The mobility maxima corresponding to the four sites of curvature discussed in the text are indicated.

FIGURE 3

(A) Typical birefringence signal, Δn, observed for the 263-bp fragment in buffer B. The dashed line corresponds to the applied pulse (arbitrary scale); the somewhat noisy line is the birefringence signal. (B) Semilogarithmic plot of the decay of the birefringence of the 263-bp fragment in buffer B as a function of time. The fractional birefringence signal, Δn(t)/Δn_o, remaining at any time, t, after the pulse is removed is plotted as a function of time. The solid line is a two-exponential fit with relaxation times of 1.36 μs (12.9%) and 8.11 μs (87.1%). (C) Semilogarithmic plot of the decay of the birefringence of the 199-bp fragment in buffer B as a function of time. The solid line is a one-exponential fit with a relaxation time of 3.74 μs. The relaxation times obtained from 15 such measurements of each sample were averaged to give the relaxation times used to calculate the τ-ratios and τ-decrements.

FIGURE 4

(A) Schematic diagram of the relationship between the four overlapping restriction fragments used to analyze the curvature of the 199-bp fragment. For ease of reference, the square boxes indicate the positions of A- or T-tracts containing five or more residues; the vertical dashed line corresponds to the location of the apparent bend center at 1922 bp. The size of each restriction fragment is indicated at the right. (B) Comparison of average τ-ratios measured in buffers B and C at 20°C with theoretical curves calculated as a function of the apparent bend angle and relative position, S, with respect to the end of the fragment. From top to bottom, the symbols correspond to fragments containing: (▵) 204 bp; (▴) 206 bp; (○) 340 bp; and (•) 199 bp. The error bar bracketing the symbol for the 204-bp fragment corresponds to the average standard deviation of the τ-ratios measured for the various fragments in buffers B and C. The values of S obtained for the 204-, 206-, 340-, and 199-bp fragments are 0.18, 0.33, 0.41, and 0.50, respectively.

SCHEME 1

SV40 sequence from 1905 to 1964 bp. The A- and T-tracts are indicated by bold letters and underlining. The position of the apparent bend center at 1922 bp is marked by a vertical arrow.

FIGURE 5

Schematic diagram of the 199-bp fragment, constructs with inserts at various positions, control fragments with sequences offset from the apparent bend center, and a 345-bp fragment with 75 bp of unbent DNA from pUC19 replacing 70 bp containing the curvature module. A- and T-tracts containing four or more residues are indicated by short vertical lines; the inserted spacers are indicated by open rectangles with widths approximately proportional to the length of the spacer, and the pUC19 substitution is indicated by a dashed rectangle. The sequences are aligned at the apparent bend center at 1922 bp, as indicated by the dotted line. Each fragment is identified in the key to the right by the total number of basepairs in the construct.

FIGURE 6

Semilogarithmic plot of DNA molecular weight, N, in basepairs, as a function of the electrophoretic mobility, μ, observed in a 6.9% T, 3% C polyacrylamide gel. (○) Normal fragments in the 50-bp ladder; (□) SV40 fragments without the curvature module or with the apparent bend center located very close to one end; (▵) insertion derivatives that are significantly curved in free solution; and (▴) insertion derivatives that are not curved, or only slightly curved, in free solution (see text). The drawn line corresponds to the fitting function describing the migration of the fragments in the 50-bp ladder. Selected fragments are identified for ease of reference.

FIGURE 7

Log-log plot of the dependence of the terminal relaxation times, τ, on DNA molecular weight, N, in bp, in buffer B at 20°C. (○) Normal control fragments; (▵) the parent 199-bp fragment, sequence mutants, and insertion derivatives; and (□) restriction fragments that do not contain the curvature module, have the apparent bend center located very close to one end, or have had the curvature module replaced by an equal-sized insert of normal DNA from pUC19. The solid line corresponds to the fitting function describing the τ-values of the normal control fragments.

FIGURE 8

Dependence of the polyacrylamide gel mobilities (μ) observed for various DNA fragments on the logarithm of the birefringence relaxation times (τ) measured in buffer C. (A) Normal DNA fragments from Figs. 6 and 7, with the calculated linear regression line. (B) The 199-bp fragment, sequence mutants, and insertion derivatives. (▵) Fragments containing inserts of various sizes; and (□) fragments that do not contain the curvature module, have the apparent bend center located very close to one end, or have had the curvature module replaced by an equal-sized insert of normal DNA from pUC19. The solid line is the regression line for the normal fragments, taken from panel A, with the data points omitted for clarity. The error bars in panels A and B represent the standard deviation of duplicate or triplicate measurements of the mobilities and relaxation times; symbols without error bars had standard deviations smaller than the size of the symbol.

FIGURE 9

Comparison of the normalized μ-decrements (−μ_decrement/N) with the average τ-decrements (<−τ_decrement>) observed for various members of the 199-bp family, using values taken from Tables 3–5 and data not shown. (○) 199-bp fragment and sequence mutants; (▿) insertion derivatives; and (□) fragments that do not contain the curvature module, have the apparent bend center located very close to one end, or have had the curvature module replaced by an equal-sized insert of normal DNA from pUC19. The parent 199-bp fragment and other fragments discussed specifically in the text are identified.