Kilkuskie R, Wood N, Ringquist S, Shinn R, Hanlon S
Department of Biological Chemistry, University of Illinois College of Medicine, Chicago 60612.
Biochemistry. 1988 Jun 14;27(12):4377-86. doi: 10.1021/bi00412a026.
Supercoiled enriched PM-2 DNA has been relaxed by treating with calf thymus topoisomerase I and used in the preparation of a family of n-butylamine adducts of varying levels of substitution. The amine is cross-linked by formaldehyde to the exocyclic amino group of G when the DNA is in duplex form. These amine adducts of covalently closed relaxed (ccr) DNA, freed of the formaldehyde and n-butylamine reactants, have circular dichroism (CD) spectral properties similar to those previously reported for the adducts of calf thymus DNA [Chen, C., Kilkuskie, R., & Hanlon, S. (1981) Biochemistry 20, 4987-4995]. In both instances, the CD transformation effected by increasing levels of substituted cationic amine is similar to that induced by solvents of high electrolyte content. The adducts also exhibit greatly increased electrophoretic mobility compared to unreacted controls or a control treated only with formaldehyde. Mobility changes in the presence of variable amounts of ethidium bromide demonstrate that this phenomenon is attributable to the formation of negative supercoils and is not due to denaturation or unwinding of the duplex. Incremental increases in superhelicity due to the attachment of the amine have been measured by reference to a topoisomerase ladder of underivatized PM-2 DNA and converted to changes in winding angle. As the extent of substitution increases, the rotational strength of the positive band above 260 nm decreases, and the winding angle increases in the nonlinear manner observed previously for underivatized PM-2 DNA [Baase, W. A., & Johnson, W. C., Jr. (1979) Nucleic Acids Res. 6, 797-814]. In fact, the relationship between these two properties is the same for both the adducts and the underivatized ccr species. Thus, the attachment of the amine has the same conformational effects as the electrolyte content of the solvent. The effect can be rationalized in terms of the reduction of the electrostatic free energy of the duplex due to site-bound or localized cation binding in the minor groove.
超螺旋富集的PM - 2 DNA已通过用小牛胸腺拓扑异构酶I处理而松弛，并用于制备一系列不同取代水平的正丁胺加合物。当DNA处于双链形式时，胺通过甲醛与G的环外氨基交联。这些共价闭合松弛（ccr）DNA的胺加合物，去除甲醛和正丁胺反应物后，具有与先前报道的小牛胸腺DNA加合物相似的圆二色性（CD）光谱特性[Chen, C., Kilkuskie, R., & Hanlon, S. (1981) Biochemistry 20, 4987 - 4995]。在这两种情况下，由取代阳离子胺水平增加引起的CD转变与高电解质含量溶剂诱导的转变相似。与未反应的对照或仅用甲醛处理的对照相比，加合物还表现出电泳迁移率大大增加。在不同量的溴化乙锭存在下的迁移率变化表明，这种现象归因于负超螺旋的形成，而不是由于双链的变性或解旋。通过参考未衍生化的PM - 2 DNA的拓扑异构酶梯来测量由于胺的附着导致的超螺旋度的增量增加，并将其转换为缠绕角的变化。随着取代程度的增加，260 nm以上正带的旋转强度降低，并且缠绕角以先前在未衍生化的PM - 2 DNA中观察到的非线性方式增加[Baase, W. A., & Johnson, W. C., Jr. (1979) Nucleic Acids Res. 6, 797 - 814]。实际上，这两种性质之间的关系对于加合物和未衍生化的ccr物种是相同的。因此，胺的附着与溶剂的电解质含量具有相同的构象效应。这种效应可以根据小沟中位点结合或局部阳离子结合导致双链静电自由能的降低来解释。
