Kilobase-range communication between polypurine.polypyrimidine tracts in linear plasmids mediated by triplex formation: a braided knot between two linear duplexes.
Kilobase-range communication between polypurine.polypyrimidine tracts in linear plasmids mediated by triplex formation: a braided knot between two linear duplexes.
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由三链体形成介导的线性质粒中聚嘌呤.聚嘧啶束之间的千碱基范围通讯：两个线性双链体之间的编织结。
作者：
K. Hampel;C. Ashley;J. S. Lee
构建了两端分别为0.34和0.94kb、相距2.8kb的两条嘧啶序列的线形载体。纤维束[d(TCCTTC)n和d(CTTCCT)n，其中n=6或12]被设计成能够彼此形成三链体，但不能与它们自己形成三链体。在精胺存在的情况下，当pH降至4时，根据琼脂糖凝胶上迁移率的变化，这些质粒形成2.8kb的分子间二聚体和分子内环。通过添加同源单链寡嘧啶而不是寡嘌呤，也可以在包含两个相同区段的线性质粒中形成系链环。在含有不同链的质粒中，加入同源单链寡嘧啶可以阻止二聚体和环的形成，但不能阻止寡嘌呤的形成。结合低pH的要求，这些结果表明三链的形成是pyr.Pur.pyr型的。二聚体和环的形成程度取决于嘧啶链的长度：在pH 6下孵育后，在含有72个碱基对(BP)插入片段的质粒中可以检测到二聚体，但在含有36个碱基对插入片段的质粒中，pH为5是必需的。滞后现象也非常明显。一旦在pH 4形成，环和二聚体在pH 8时无限期保持稳定，这表明结构在拓扑上被捕获。然而，通过与核酸酶P1孵育，这些结构被分解成组分线状质粒。这是首次展示了两个线性双链之间的编织或氢键打结，并可能对染色体环的形成产生影响。
Linear plasmids were constructed containing two pyrimidine tracts that were 0.34 and 0.94 kilobases (kb) from either end and were separated by 2.8 kb. The tracts [d(TCCTTC)n and d(CTTCCT)n where n = 6 or 12] were designed so as to be able to form triplexes with each other but not with themselves. Upon lowering of the pH to 4 in the presence of spermine, these plasmids form intermolecular dimers and intramolecular loops of 2.8 kb, as judged from mobility changes on agarose gels. A tethered loop could also be formed in a linear plasmid containing two identical tracts by adding an homologous single-stranded oligopyrimidine, but not an oligopurine. In plasmids containing different tracts, the formation of both dimers and loops could be blocked by adding either homologous single-stranded oligopyrimidine but not an oligopurine. Together with the requirement of low pH, these results demonstrate that triplex formation is of the pyr.pur.pyr type. The extent of dimer and loop formation was dependent on the length of the pyrimidine tract: dimers could be detected in plasmids containing the 72 base pair (bp) inserts after incubation at pH 6, but in plasmids containing the 36 bp inserts, a pH of 5 was required. Hysteresis was also evident to a remarkable extent. Once formed at pH 4, loops and dimers remained stable indefinitely at pH 8, suggesting that the structures become topologically trapped. However, the structures were resolved into the component linear plasmids by incubation with nuclease P1. This is the first demonstration of a braided or hydrogen-bonded knot between two linear duplexes and may have implications for chromosomal loop formation.
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