Yongjun Lu

Curved DNA molecules migrate anomalously slowly in free solution

The electrophoretic mobility of a curved DNA restriction fragment taken from the VP1 gene in the SV40 minichromosome has been measured in polyacrylamide gels and free solution, using capillary electrophoresis. The 199 bp restriction fragment has an apparent bend angle of 46 ± 2° located at SV40 sequence position 1922 ± 2 bp [Lu Y.J., Weers B.D. and Stellwagen N. C. (2005) Biophys. J., 88, 1191–1206]. The ‘curvature module’ surrounding the apparent bend center contains five unevenly spaced A- and T-tracts, which are responsible for the observed curvature. The parent 199 bp fragment and sequence mutants containing at least one A-tract in the curvature module migrate anomalously slowly in free solution, as well as in polyacrylamide gels. Hence, the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are due in part to their anomalously slow mobilities in free solution. Analysis of the gel and free solution mobility decrements indicates that each A- or T-tract contributes independently, but not equally, to the curvature of the 199 bp fragment and its A-tract mutants. The relative contribution of each A- or T-tract to the observed curvature depends on its spacing with respect to the first A-tract in the curvature module.

Analysis of the intrinsic bend in the M13 origin of replication by atomic force microscopy.

Atomic force microscopy (AFM) has been used to image a 471-bp bent DNA restriction fragment derived from the M13 origin of replication in plasmid LITMUS 28, and a 476-bp normal, unbent fragment from plasmid pUC19. The most probable angle of curvature of the 471-bp DNA fragment is 40-50 degrees, in reasonably good agreement with the bend angle determined by transient electric birefringence, 38 degrees +/- 7 degrees. The normal 476-bp DNA fragment exhibited a Gaussian distribution of bend angles centered at 0 degrees, indicating that this fragment does not contain an intrinsic bend. The persistence length, P, was estimated to be 60 +/- 8 and 62 +/- 8 nm for the 471- and 476-bp fragments, respectively, from the observed mean-square end-to-end distances in the AFM images. Since the P-values of the normal and bent fragments are close to each other, the overall flexibility of DNA fragments of this size is only marginally affected by the presence of a stable bend. The close agreement of AFM and transient electric birefringence results validates the suitability of both methods for characterizing DNA bending and flexibility.