Lu Yongjun, Stellwagen Nancy C
Department of Biochemistry, University of Iowa, Iowa City, Iowa.
Biophys J. 2008 Mar 1;94(5):1719-25. doi: 10.1529/biophysj.107.121236. Epub 2007 Nov 9.
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.
已经测定了由DNA A序列(四个或更多连续腺嘌呤或胸腺嘧啶残基的序列)对两个弯曲的约200个碱基对(bp)的限制性片段的单价阳离子结合情况,其中一个片段取自M13复制起点,另一个取自SV40的VP1基因。先前已表明这两个片段分别在包含四个或五个不规则间隔A序列的约60 bp“曲率模块”内含有稳定的、位于中心的44度和46度弯曲。对这两个片段、SV40曲率模块中A序列数量减少的序列变体或M13曲率模块中A序列侧翼残基发生变化的序列变体进行的瞬态电双折射测量,已与使用已知方程的相同片段的自由溶液电泳迁移率相结合,以估计每个片段的有效电荷。曲率模块中的每个A序列平均使有效电荷减少三分之一,这表明每个A序列大约有三分之一的时间结合一个单价阳离子。需要单价阳离子与两个或更多A序列结合才能观察到DNA螺旋轴的明显弯曲。