Tn10, like several other transposons, exhibits a marked preference for integration into particular target sequences. Such sequences are referred to as integration hotspots and have been used to define a consensus target site in Tn10 transposition. We demonstrate that a Tn10 hotspot called HisG1, which was identified originally in vivo, also functions as an integration hotspot in vitro in a reaction where the HisG1 sequence is present on a short DNA oligomer. We use this in vitro system to define factors which are important for the capture of the HisG1 target site. We demonstrate that although divalent metal ions are not essential for HisG1 target capture, they greatly facilitate capture of a mutated HisG1 site. Analysis of catalytic transposase mutants further demonstrates that the DDE motif plays a critical role in 'divalent metal ion-dependent' target capture. Analysis of two other classes of transposase mutants, Exc+ Int- (which carry out transposon excision but not integration) and ATS (altered target specificity), demonstrates that while a particular ATS transposase binds HisG1 mutants better than wild-type transposase, Exc+ Int- mutants are defective in HisG1 capture, further defining the properties of these classes of mutants. Possible mechanisms for the above observations are considered.

译文

像其他几种转座子一样,Tn10表现出明显的整合特定靶序列的偏好。此类序列称为整合热点,已用于定义Tn10转座中的共有靶位点。我们证明了一个称为HisG1的Tn10热点,最初在体内被发现,在一个短DNA低聚物上存在HisG1序列的反应中,它在体外还起着整合热点的作用。我们使用这个体外系统来定义对于捕获HisG1目标位点很重要的因素。我们证明,尽管二价金属离子对于HisG1目标捕获不是必需的,但它们极大地促进了突变的HisG1位点的捕获。催化转座酶突变体的分析进一步表明,DDE基序在“二价金属离子依赖性”靶标捕获中起关键作用。分析另外两类转座酶突变体Exc Int-(进行转座子切除但不整合)和ATS(改变的靶标特异性)表明,虽然特定的ATS转座酶与HisG1突变体的结合比野生型转座酶Exc Int-突变体在HisG1捕获中存在缺陷,进一步定义了这类突变体的特性。考虑了上述观察的可能机制。

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