Intrinsic Curvature in the VP1 Gene of SV40: Comparison of solution and gel results
Yongjun Lu 1, Brock D. Weers 2 and Nancy C. Stellwagen 1*
1 University of Iowa
2 Texas A & M University
* To whom correspondence should be addressed. E-mail: nancy-stellwagen{at}uiowa.edu.
Submitted on January 12, 2004
Revised on March 1, 2004
Accepted on 29 October 2004
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Abstract |
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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 base pairs. 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 report, the results observed in free solution and in polyacrylamide gels are compared for a highly curved 199 base pair 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 46º ± 2º centered at sequence position 1922 ± 2 bp. Four unphased A- and T-tracts and a mixed A3T4-tract occur within a span of ~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 fragment 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. The 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.
Key Words:
DNA curvature, SV40, anomalous electrophoretic mobilities, circular permutation assay, transient electric birefringence
