Correct integration of model substrates by Ty1 integrase.
Correct integration of model substrates by Ty1 integrase.
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Ty1 整合酶正确整合模型底物。
DOI：
10.1128/jvi.74.24.11522-11530.2000
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发表时间：
2000
影响因子：
5.4
通讯作者：
Garfinkel,DJ
中科院分区：
文献类型：
--
作者：
Moore,SP;Garfinkel,DJ
酿酒酵母（Saccharomycescerevisiae）的逆转录病毒样移动的遗传元件Ty1通过元件编码的整合酶（IN）转移到新的基因组位置。在这里，我们报告说，纯化的重组IN催化正确整合的线性DNA到一个超螺旋的靶质粒。Ty1病毒样颗粒（VLP）比IN更有效地整合供体DNA。VLP和IN介导的插入发生在靶中的随机位点。Mg 2+优于Mn 2+的正确整合，和阳离子增强非特异性核酸酶活性的IN。通过Southern分析也获得了与正确整合事件一致的产物。重组IN和VLP利用了许多但不是全部的含有非Ty1末端的线性供体片段，包括已被证明在体内转座有缺陷的U3突变。总之，我们的研究结果表明，IN是足够的Ty1整合在体外和IN与外源性捐助者的相互作用不太严格，比与内源性元素。
The retrovirus-like mobile genetic element ofSaccharomyces cerevisiae, Ty1, transposes to new genomic locations via the element-encoded integrase (IN). Here we report that purified recombinant IN catalyzed correct integration of a linear DNA into a supercoiled target plasmid. Ty1 virus-like particles (VLPs) integrated donor DNA more efficiently than IN. VLP and IN-mediated insertions occurred at random sites in the target. Mg2+was preferred over Mn2+for correct integration, and neither cation enhanced nonspecific nuclease activity of IN. Products consistent with correct integration events were also obtained by Southern analysis. Recombinant IN and VLPs utilized many, but not all, linear donor fragments containing non-Ty1 ends, including a U3 mutation which has been shown to be defective for transposition in vivo. Together, our results suggest that IN is sufficient for Ty1 integration in vitro and IN interacts with exogenous donors less stringently than with endogenous elements.