Bianchi A, Wells R D, Heintz N H, Caddle M S
Department of Biochemistry, School of Medicine, University of Alabama, Birmingham 35294.
J Biol Chem. 1990 Dec 15;265(35):21789-96.
The earliest replicating portion of the Chinese hamster dihydrofolate reductase domain contains a cluster of simple repeated sequences 180 base pairs long composed of 5'-(GC)5(AC)18(AG)21(G)9(CAGA)4GAGGGAGAGAGGCAGAGAGGG(AG)27-3 '. Previous nuclease sensitivity and intermolecular hybridization studies suggested that the two long (AG) repeats in this tract formed intramolecular DNA triplexes in negatively supercoiled plasmids at pH 5.2 (Caddle, M. S., Lussier, R. L., and Heintz, N. H. (1990) J. Mol. Biol. 211, 19-33). To further characterize the structural organization, supercoiled plasmids containing this region were analyzed in vitro with OsO4 and diethyl pyrocarbonate probes as well as with two-dimensional gel electrophoresis under different conditions. In pMCG, which contains the sequence in a 1.6-kilobase pair insert, the preferred conformation at neutral pH and at the native superhelical density is a Z-DNA structure for the (GC)5(AC)18 tract. Under mildly acidic conditions and at the native superhelical density, both (AG) tracts form intramolecular triplexes to the exclusion of the Z-DNA structure. Chemical probing of topoisomers of pMCG indicates that the (AG)27 tract forms a triplex more readily than the (AG)21 motif. Also, analysis of the reactivity obtained on a larger plasmid, pMCD, which contains the cluster of repeated sequences in a 4.75-kilobase pair insert, shows that at the native superhelical density the formation of intramolecular triplexes is limited to the (AG)27 tract. Finally, experiments conducted on different populations of topoisomers of pMCG show the existence, at pH 5.0 and highly negative superhelical density (greater than or equal to 0.080), of both the left-handed and the two triple-stranded structures in the same DNA. Therefore, one triplex is located immediately adjacent to the Z helix. Companion studies revealed that this region of the DHFR replicon modulates fork translocation during the replication of recombinant plasmids in mammalian cells.
中国仓鼠二氢叶酸还原酶结构域最早进行复制的部分包含一段由180个碱基对组成的简单重复序列簇，其序列为5'-(GC)5(AC)18(AG)21(G)9(CAGA)4GAGGGAGAGAGGCAGAGAGGG(AG)27-3'。先前的核酸酶敏感性和分子间杂交研究表明，在pH 5.2的负超螺旋质粒中，该区域的两个长(AG)重复序列形成了分子内DNA三链体（卡德尔，M. S.，卢西尔，R. L.，和海因茨，N. H.（1990年）《分子生物学杂志》211卷，19 - 33页）。为了进一步表征其结构组织，使用四氧化锇和焦碳酸二乙酯探针以及在不同条件下的二维凝胶电泳对含有该区域的超螺旋质粒进行了体外分析。在pMCG中，该序列包含在一个1.6千碱基对的插入片段中，在中性pH和天然超螺旋密度下，(GC)5(AC)18区域的首选构象是Z-DNA结构。在轻度酸性条件和天然超螺旋密度下，两个(AG)区域都形成分子内三链体，从而排除了Z-DNA结构。对pMCG拓扑异构体的化学探测表明，(AG)27区域比(AG)21基序更容易形成三链体。此外，对更大的质粒pMCD进行的反应性分析表明，该质粒的4.75千碱基对插入片段中包含重复序列簇，在天然超螺旋密度下，分子内三链体的形成仅限于(AG)27区域。最后，对pMCG不同拓扑异构体群体进行的实验表明，在pH 5.0和高度负超螺旋密度（大于或等于0.080）下，同一DNA中同时存在左手螺旋结构和两种三链结构。因此，一个三链体紧邻Z螺旋。相关研究表明，DHFR复制子的这一区域在重组质粒在哺乳动物细胞中的复制过程中调节叉的移位。
