MECHANISMS OF RETROTRANSPOSON REPLICATION

含有LTR的逆转录转座子和逆转录病毒代表了两种 逆转录酶系统进化树上的相关分支 （RT）-含有移动的遗传元件。这两种类型的元素都使用 RNA依赖的DNA聚合酶，RT，复制他们的RNA基因组成双 然后将其插入宿主基因组中。的移动 基因组内的逆转录转座子可能是有害的，导致基因组 不稳定、癌症、不育和突变。另一方面 反转录转座子积极地促进宿主基因组的进化。 我们长期关注的是 反转录转座子Tyl维持自身作为移动的内源家族 其宿主生物酿酒酵母中的元素。泰勒是一个特别 有用的实验系统来研究换位，因为它是如此容易 操纵，因为它的天然宿主是这样一个遗传上易于控制的有机体， 因为它的复制步骤与 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， 它们的合成的保真度是它们的生物化学决定因素。 功能能力。我们已经发现Tyl RT是一种易错聚合酶， 它通过一种新的机制在特定位点产生错误。我们建议（目标 1)从遗传学和生物化学上探索这种新的诱变机制， 确定这一过程对Tyl复制适应性的影响。此外，本发明还 我们将确定和表征其他诱变机制所采用的 复制期间的Tyl RT。我们创作了一部小说 Tyl RT的活性位点突变，其仍然可以合成DNA，但 阻止了换位。这种缺陷可以通过两个基因的突变来抑制， 我们计划（目的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， 复制的我们将进一步确定这部小说的性质是否 突变体可推广到普遍存在的逆转录转座子家族， 以前没有被研究过。

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.