Palecek E, Boubliková P, Nejedlý K, Galazka G, Klysik J
Institute of Biophysics, Czechoslovak Academy of Sciences, Brno.
J Biomol Struct Dyn. 1987 Oct;5(2):297-306. doi: 10.1080/07391102.1987.10506395.
Structural distortions on the boundary between right-handed and left-handed DNA segments in negatively supercoiled plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of osmium tetroxide, pyridine and glyoxal. These two probes react preferentially with single-stranded DNA, but only the latter requires non-paired bases for the reaction. Nuclease S1 and testing of the inhibition of BamHI cleavage (whose recognition sequences GGATCC lie on the "outer" boundaries between the (dC-dG)n and the pBR322 nucleotide sequence) were used to detect the site-specific chemical modification in pRW751. As a result of glyoxal treatment BamHI cleavage was strongly inhibited in topoisomeric samples whose superhelical density was sufficiently negative to stabilize the (dC-dG)n segments in the left-handed form. Osmium tetroxide, pyridine modification resulted in a similar inhibition of BamHI cleavage and in a formation of nuclease S1 sensitive sites. The results suggest that the "outer" B-Z junctions in pRW751 contain one or few non-paired bases or non-Watson-Crick base pairs.
利用四氧化锇、吡啶和乙二醛研究了负超螺旋质粒pRW751（pBR322的衍生物，含有(dC-dG)13和(dC-dG)16片段）中右手和左手DNA片段边界处的结构畸变。这两种探针优先与单链DNA反应，但只有后者反应需要未配对碱基。使用核酸酶S1以及测试BamHI切割的抑制情况（其识别序列GGATCC位于(dC-dG)n与pBR322核苷酸序列之间的“外部”边界上）来检测pRW751中的位点特异性化学修饰。乙二醛处理的结果是，在拓扑异构体样品中，当超螺旋密度足够负以稳定左手形式的(dC-dG)n片段时，BamHI切割受到强烈抑制。四氧化锇、吡啶修饰导致类似的BamHI切割抑制，并形成核酸酶S1敏感位点。结果表明，pRW751中的“外部”B-Z连接包含一个或几个未配对碱基或非沃森-克里克碱基对。
