Poly d(GC)在负超螺旋下形成的Z-DNA

Formation of Z-DNA in Poly d(GC) in the Presence of Negatively Supercoil

为建立一个生理状态下的左旋DNA(Z-DNA)模型,本文以pMD18-T质粒为母本质粒,将含有d(GC)_6、d(GC)_8、d(GC)_(10)、d(GC)_(13)等poly d(GC)片段的特殊序列插入到pMD18-T质粒中,构建了poly d(GC)重组质粒。这些poly d(GC)重组质粒处于负超螺旋状态。甲基化抑制实验证实,这些插入到质粒中的poly d(GC)能够形成潜在的Z-DNA构象。同时,为进一步验证poly d(GC)重复的长度与形成Z-DNA效率的关系,原核表达并亲和层析纯化了鲫鱼PKZZα多肽(P★)。凝胶阻滞实验分析了d(GC)_6、d(GC)_8、d(GC)_(10)、d(GC)_(13)4种重组质粒与P_(Zα)的亲和性。结果显示,P_(Zα)对这4种重组质粒的迁移都能产生阻滞效应,并且随着GC的增多,阻滞效应越明显。说明d(GC)越多,poly d(GC)形成Z-DNA的能力越强。

In order to build a Z-DNA model in a physiological status, this study took the plasmid pMD 18-T as the parental plasmid. Some special sequences consisting of such poly d（GC） segments as d（GC）6, d（GC）8, d（GC）10 and d（GC）13 were inserted into the plasmid pMD 18-T, so as to form recombinant plasmid/poly d（GC）. These recombinant plasmid/poly d（GC） were in a state of negatively supercoil. The poly d（GC） which was inserted into the plasmid can form the latent Z-DNA proved by methylate inhibitory experiment. Besides, in order to prove the relation between the length of poly d（GC） and the efficiency of forming Z-DNA. Pzα was expressed by a prokaryotic expression system and then purified by affinity chromatography. The affinity of Pzα peptides with the 4 recombinant plasmids d（GC）6, d（GC）8, d（GC）10 and d（GC）13 was analyzed by Bandshift assay. The result showed that Pzα can generate the retardation effects on the transfer of the 4 recombinant plasmids. The more GC they have, the more obvious the retardation effects are. This indicates that the ability of poly d（GC） forming Z-DNA depends on the number of d（GC） repeats.