MECHANISMS OF RETROTRANSPOSON REPLICATION

含ltr的逆转录转座子和逆转录病毒代表两种 逆转录酶系统发育树上的相关分支 (RT)-含有可移动的遗传元件。两种类型的元素都使用 依赖于RNA的DNA聚合酶，RT，将它们的RNA基因组复制成双倍 链DNA，然后插入到宿主基因组中。运动的运动 基因组中的反转录转座子可能是有害的，导致基因组 不稳定、癌症、不孕不育和突变。另一方面， 反转录转座子积极促进其宿主基因组的进化。 我们对使用的复制机制具有长期兴趣 反转录转座子TYL维持其作为内源性移动蛋白家族的地位 其寄主酿酒酵母中的元素。泰尔是一个特别的 用于研究换位的有用的实验系统，因为它非常容易 被操纵，因为它的自然宿主是这样一种遗传上易驯服的有机体， 因为它的复制中的步骤与这些步骤密切相关 参与逆转录病毒复制。 由于DNA聚合酶复制和传递重要的遗传信息， 它们合成的保真度是它们的关键生化决定因素 功能能力。我们发现TYL RT是一种容易出错的聚合酶， 它通过一种新的机制在特定的位置产生错误。我们建议(目的) 1)从遗传和生化角度探讨这一新的诱变机制； 确定这一过程对TYL复制适合性的影响。此外， 我们将确定和表征其他使用的突变机制 复制期间的TYL RT。我们已经创作了一部小说，并正在对其进行描述 Tyl RT活性部位突变，仍可合成DNA，但 因移位而被封堵。这一缺陷可以通过两个基因的突变来抑制 RT的核糖核酸酶H和聚合酶结构域。我们计划(目标2)使用这种新的突变体 研究聚合和复制之间的区别，以及更多 广泛地说，为了了解TYL过程中蛋白质结构域之间的相互作用 复制。此外，我们将确定这部小说的属性是否 突变体是普遍存在的反转录转座子家族，其RTS 以前都没有被研究过。

LTR-containing retrotransposons and retroviruses represent two related branches on the phylogenetic tree of reverse transcriptase (RT)-containing mobile genetic elements. Both types of elements use the RNA-dependent DNA polymerase, RT, to replicate their RNA genomes into double strand DNA which is then inserted into a host genome. The movement of retrotransposons within a genome can be harmful, resulting in genomic instability, cancer, sterility and mutations. On the other hand retrotransposons actively contribute to the evolution of their hosts' genomes. We have a long term interest in the replication mechanisms utilized by retrotransposon Tyl to maintain itself as an endogenous family of mobile elements in its host organism Saccharomyces cerevisiae. Tyl is a particularly useful experimental system to study transposition because it is so easily manipulated, because its natural host is such a genetically tractable organism, and because the steps in its replication are so closely related to the steps involved in retroviral replication. Since DNA polymerases replicate and transmit vital genetic information, the fidelity of their synthesis is a crucial biochemical determinant of their functional capacity. We have found that Tyl RT is an error prone polymerase, that generates errors at specific sites by a novel mechanism. We propose (Aim 1) to explore this new mutagenic mechanism genetically and biochemically, and determine the consequences of this process to Tyl replication fitness. Further, we will identify and characterize additional mutagenic mechanisms employed by Tyl RT during replication. We have created and are characterizing a novel mutation in the active site of Tyl RT which can still synthesize DNA but is blocked for transposition. This defect is suppressible by mutations in both the RNase H and polymerase domains of RT. We plan (Aim 2) to use this novel mutant to examine the distinctions between polymerization and replication, and more broadly, to understand the interactions between protein domains during Tyl replication. Further we will determine whether the properties of this novel mutant are generalizable to the ubiquitous family of retrotransposons whose RTs have not previously been studied.