Stellwagen Earle, Lu Yongjun, Stellwagen Nancy C
Department of Biochemistry, University of Iowa, Iowa City, IA, USA.
Nucleic Acids Res. 2005 Aug 5;33(14):4425-32. doi: 10.1093/nar/gki748. Print 2005.
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 degrees 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.
利用毛细管电泳技术,在聚丙烯酰胺凝胶和自由溶液中测量了从SV40微型染色体的VP1基因中获取的弯曲DNA限制片段的电泳迁移率。这个199bp的限制片段在SV40序列位置1922±2bp处有一个明显的弯曲角度,为46±2度[Lu Y.J., Weers B.D.和Stellwagen N.C.(2005年),《生物物理杂志》,88卷,1191 - 1206页]。围绕明显弯曲中心的“曲率模块”包含五个间距不均的A - 序列和T - 序列,这导致了观察到的弯曲。亲本199bp片段以及在曲率模块中包含至少一个A - 序列的序列突变体,在自由溶液以及聚丙烯酰胺凝胶中迁移异常缓慢。因此,在聚丙烯酰胺凝胶中观察到的弯曲DNA分子异常缓慢的迁移率,部分归因于它们在自由溶液中异常缓慢的迁移率。对凝胶和自由溶液迁移率降低的分析表明,每个A - 序列或T - 序列对199bp片段及其A - 序列突变体的曲率有独立但不均等的贡献。每个A - 序列或T - 序列对观察到的曲率的相对贡献,取决于它相对于曲率模块中第一个A - 序列的间距。