Noronha Anne M, Noll David M, Wilds Christopher J, Miller Paul S
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205, USA.
Biochemistry. 2002 Jan 22;41(3):760-71. doi: 10.1021/bi011610u.
The preparation and physical properties of short DNA duplexes that contain a N(4)C-ethyl-N(4)C interstrand cross-link are described. Duplexes that contain an interstrand cross-link between mismatched C-C residues and duplexes in which the C residues of a -CG- or -GC- step are linked to give "staggered" interstrand cross-links were prepared using a novel N(4)C-ethyl-N(4)C phosphoramidite reagent. Duplexes with the C-C mismatch cross-link have UV thermal transition temperatures that are 25 degrees C higher than the melting temperatures of control duplexes in which the cross-link is replaced with a G-C base pair. It appears that this cross-link stabilizes adjacent base pairs and does not perturb the structure of the helix, a conclusion that is supported by the CD spectrum of this duplex and by molecular models. An even higher level of stabilization, 49 degrees C, is seen with the duplex that contains a -CG- staggered cross-link. Molecular models suggest that this cross-link may induce propeller twisting in the cross-linked base pairs, and the CD spectrum of this duplex exhibits an unusual negative band at 298 nm, although the remainder of the spectrum is similar to that of B-form DNA. Mismatched C-C or -CG- staggered cross-linked duplexes that have complementary overhanging ends can undergo self-ligation catalyzed by T4 DNA ligase. Analysis of the ligated oligomers by nondenaturing polyacrylamide gel electrophoresis shows that the resulting oligomers migrate in a manner similar to that of a mixture of non-cross-linked control oligomers and suggests that these cross-links do not result in significant bending of the helix. However, the orientation of the staggered cross-link can have a significant effect on the structure and stability of the cross-linked duplex. Thus, the thermal stability of the duplex that contains a -GC- staggered cross-link is 10 degrees C lower than the melting temperature of the control, non-cross-linked duplex. Unlike the -CG- staggered cross-link, in which the cross-linked base pairs can still maintain hydrogen bond contacts, molecular models suggest that formation of the -GC- staggered cross-link disrupts hydrogen bonding and may also perturb adjacent base pairs leading to an overall reduction in helix stability. Duplexes with specifically positioned and oriented cross-links can be used as substrates to study DNA repair mechanisms.
描述了含有N(4)C - 乙基 - N(4)C链间交联的短DNA双链体的制备及其物理性质。使用新型的N(4)C - 乙基 - N(4)C亚磷酰胺试剂制备了在错配的C - C残基之间含有链间交联的双链体，以及其中 - CG - 或 - GC - 步的C残基相连形成“交错”链间交联的双链体。具有C - C错配交联的双链体的紫外热转变温度比用G - C碱基对取代交联的对照双链体的解链温度高25℃。似乎这种交联稳定了相邻的碱基对并且不会扰乱螺旋结构，这一结论得到了该双链体的圆二色光谱和分子模型的支持。对于含有 - CG - 交错交联的双链体，观察到更高水平的稳定性，为49℃。分子模型表明这种交联可能会在交联的碱基对中诱导螺旋桨扭曲，并且该双链体的圆二色光谱在298nm处呈现出异常的负带，尽管光谱的其余部分与B型DNA相似。具有互补突出端的错配C - C或 - CG - 交错交联双链体可以在T4 DNA连接酶催化下进行自连接。通过非变性聚丙烯酰胺凝胶电泳对连接的寡聚物进行分析表明，所得寡聚物的迁移方式类似于非交联对照寡聚物混合物的迁移方式，这表明这些交联不会导致螺旋的显著弯曲。然而，交错交联的方向对交联双链体的结构和稳定性有显著影响。因此，含有 - GC - 交错交联的双链体的热稳定性比对照非交联双链体的解链温度低10℃。与交联碱基对仍可保持氢键接触的 - CG - 交错交联不同，分子模型表明 - GC - 交错交联的形成会破坏氢键，还可能扰乱相邻的碱基对，导致螺旋稳定性整体降低。具有特定定位和取向交联的双链体可作为研究DNA修复机制的底物。
