DNA双链区的切割导致碱变性超螺旋质粒pBR322的分子内结节（英文）  

Cleavage of The Double-strand Region in Alkali-denatured Supercoiled Plasmid pBR322 Leads to DNA Nodes

作　　者：田磊[1] 苏小运[2] 张臻峰[3] 黄熙泰[4] 

机构地区：[1]中国人民解放军海军总医院普外科,北京100048 [2]中国农业科学院饲料研究所,北京100081 [3]中国科学院微生物研究所微生物资源前期开发国家重点实验室,北京100101 [4]南开大学生命科学学院,天津300071

出　　处：《生物化学与生物物理进展》2016年第8期778-786,共9页Progress In Biochemistry and Biophysics

基　　金：supported by grants from The National Natural Science Foundation of China(31270124,30900023)~~

摘　　要：为进一步研究碱变性超螺旋D NA(Ⅳ型D NA;DNAⅣ)的结构,对碱变性质粒p BR322进行了酶学分析并利用原子力显微镜(AFM)对新形成的D NA结构进行了观察和比较.在所有已检测的限制酶中只有PstⅠ可以切割质粒p BR322的Ⅳ型DNA分子,说明在这种变性的DNA分子中仍存在少数完整的限制酶识别位点.与碱变性DNA分子的闭合环状结构相反,AFM成像的结果显示PstⅠ处理后的DNAⅣ分子均为开放结构,同时这种分子包含明显的DNA结节.与DNAⅣ分子相比,这种D NA分子的表观长度缩短了大约11%.有意思的是,大肠杆菌拓扑异构酶Ⅳ(一种Ⅱ型拓扑异构酶)也可以在p BR322 DNAⅣ分子中引入分子内结节,而这种结节D NA分子仍然保持闭合状态.AFM的结果表明上述两种结节的DNA分子在表观长度及结节结构的尺寸上均比较相似.这些发现证实,在碱变性的超螺旋DNA分子中仍然保留着一些长度较短的、含有特异性碱基配对的D NA双链区,而在这些区域内的D NA双链断裂可以导致D NA结节.Combinatorial analyses including enzymatic analysis and atomic force microscopy（AFM） were used to gain more insights into structure of the alkali-denatured plasmid p BR322 DNA（DNA Ⅳ）.Among the restriction enzymes tested,PstⅠ was able to cleave the p BR322 DNA Ⅳ molecules,indicative of the existence of an intact restriction site in the denatured DNA.AFM images revealed that PstⅠ-treated DNA Ⅳ molecules contained an individual node.The contour length of the condensed molecules was decreased by about 11% compared to DNA Ⅳ.Intriguingly,the Escherichia coli topoisomerase Ⅳ,a type Ⅱ topoisomerase,also introduced nodes into DNA Ⅳ while keeping the molecules in a closed form.The two types of DNA were similar to each other in terms of the contour length and the size of the node based on the AFM analysis.These findings suggest that specific base-paired regions exist in DNA Ⅳ,and that cleavages at these regions may lead to DNA node.

关 键 词：DNAⅣ 限制性内切酶 DNA拓扑异构酶 DNA结节 原子力显微镜 

分 类 号：Q5[生物学—生物化学]