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首页> 外文期刊>Journal of Molecular Biology >Shope fibroma virus DNA topoisomerase catalyses holliday junction resolution and hairpin formation in vitro.
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Shope fibroma virus DNA topoisomerase catalyses holliday junction resolution and hairpin formation in vitro.

机译：Shope纤维瘤病毒DNA拓扑异构酶在体外催化霍利迪结的分辨和发夹的形成。

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The telomeres of poxviral chromosomes comprise covalently closed hairpin structures bearing mismatched bases. These hairpins are formed as concatemeric replication intermediates and are processed into mature, unit-length genomes. The structural transitions and enzymes involved in telomere resolution are poorly understood. Here we show that the type I topoisomerase of Shope fibroma virus (SFV) can promote a recombination reaction which converts cloned SFV replication intermediates into hairpin-ended molecules resembling mature poxviral telomeres. Recombinant SFV topoisomerase linearised a palindromic plasmid bearing 1.5 kb of DNA encoding the SFV concatemer junction, at a site near the centre of inverted-repeat symmetry. Most of these linear reaction products bore hairpin tips as judged by denaturing gel electrophoresis. The resolution reaction required palindromic SFV DNA sequences and was inhibited by compounds which block branch migration (MgCl2) or poxviral topoisomerases. The resolution reaction was also slow, needed substantial quantities of topoisomerase, and required that the palindrome be extruded in a cruciform configuration. DNA cleavage experiments identified a pair of suitably oriented topoisomerase recognition sites, 90 bases from the centre of the cloned SFV terminal inverted repeat, which may mark the resolution site. These data suggest a resolution scheme in which branch migration of a Holliday junction through a site occupied by covalently bound topoisomerase molecules, could lead to telomere resolution. Copyright 1999 Academic Press.
机译：痘病毒染色体的端粒包含带有不匹配碱基的共价闭合的发夹结构。这些发夹形成为串联的复制中间体，并加工成成熟的单位长度基因组。端粒拆分涉及的结构转变和酶了解甚少。在这里，我们显示Shope纤维瘤病毒（SFV）的I型拓扑异构酶可以促进重组反应，该重组反应将克隆的SFV复制中间体转化为类似于成熟痘病毒端粒的发夹末端分子。重组SFV拓扑异构酶在一个反向重复对称中心附近的位点上线性化了一个回文质粒，该质粒含有1.5 kb的编码SFV串联连接的DNA。通过变性凝胶电泳判断，大多数这些线性反应产物带有发夹尖端。拆分反应需要回文SFV DNA序列，并被阻断分支迁移（MgCl2）或痘病毒拓扑异构酶的化合物抑制。拆分反应也很慢，需要大量的拓扑异构酶，并且需要将回文状以十字形挤出。 DNA切割实验确定了一对适当定向的拓扑异构酶识别位点，距离克隆的SFV末端反向重复序列中心90个碱基，可能标记了分离位点。这些数据提出了一个解决方案，其中霍利迪结的分支迁移通过共价结合的拓扑异构酶分子占据的位点，可能导致端粒分离。版权所有1999，学术出版社。

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