Ty Element Retrotransposition in Saccharomyces cerevisiae

我们的研究涉及Ty元件逆转录转座的机制和后果 在芽殖酵母中。Ty元素包括 通过RNA转座的长末端重复序列（LTR）反转录转座子的五个相关家族 中间体Ty基因组包含两个基因，对应于 Gag和Pol基因 逆转录病毒逆转录转座子被转录成基因组长度的RNA，这是 用于通过元件编码逆转录酶蛋白进行逆转录的模板， 翻译. Ty蛋白成熟和逆转录发生在Ty病毒样 颗粒（Ty-VLP），这似乎是必不可少的转座过程。虽然 Ty-VLP在细胞质中积累，这是一种含有Ty cDNA的Ty整合前复合物， 元件编码的整合酶和其他蛋白质必须通过核膜才能获得 获取基因组每个Ty元素类非随机积分并具有不同的 受染色质状态或RNA聚合酶III影响的靶向机制 转录因子所有可用的证据表明，Ty元素仍然存在于细胞内 并且不具有传染性。因此，这些元素及其宿主已经进化出控制力 将转座和元件介导的基因组重排保持在低水平的机制， 以及整合位点偏好，其降低了引起有害突变的可能性。 在过去的一年里，我们 已经在表征调节Ty 1反转录转座的宿主基因方面取得了进展。的 第一项研究涉及4739个基因缺失突变体的系统筛选，以确定那些 增加Ty 1迁移率（Ty 1限制或RTT基因）。之间 鉴定的91个突变体中，80%编码参与核过程的产物，如染色质 结构和功能，DNA修复和重组，以及转录。然而，在这方面， 包括额外Ty 1和Ty 3筛选的生物信息学分析表明， 参与多种生物过程的基因影响Ty在酵母中的迁移率。进一步 我们鉴定了33个RTT突变体， 筛选显示Ty 1 RNA水平在5个突变体中增加，其余影响迁移率 转录后的Ty 1 RNA和cDNA水平在缺陷突变体中保持不变， 转录延伸，包括ckb 2和 elf 1，表明Ty 1整合可能更多 在这些菌株中有效。插入位点偏好 CAN 1基因座需要Ty 1限制性基因参与 组蛋白H2 B通过Paf复合物亚基基因的泛素化，以及 BRE 1和RAD 6，组蛋白 通过RTT 109的H3乙酰化，和 ASF 1和转录延长， SPT 5。我们的研究结果表明，多个途径限制 Ty 1迁移率和组蛋白修饰可以保护编码区免受插入 诱变由于这些基因也是RNA聚合酶有效转录所必需的， II，Ty 1插入的额外靶点可能被停滞的转录复合物所发现。 正在进行的工作集中在定义Ty 1整合酶靶向结构域和理解其功能。 野生型和靶向缺陷型中可用于转座事件的基因组景观 变种人考虑 在各种筛选中鉴定的大量调节Ty逆转录转座的基因， 我们考虑了很多RTT基因 通过几个共同的途径行动。支持这一观点的证据来自最近一项关于 Ty 1限制性基因的子集，与Joan Curcio的实验室合作进行 （纽约州奥尔巴尼的沃兹沃斯中心）。Ty 1在芽殖酵母中的移动性受到一系列 在DNA复制过程中保持基因组完整性的蛋白质， 修复.然而，参与增加Ty 1 cDNA水平和细胞迁移率的机制尚不清楚。 缺乏这些Rtt因子，其中一些是哺乳动物逆转录病毒的直系同源物 限制性因素的特征很差。有趣的是，两个S期检查点通路， 复制应激途径或DNA损伤途径，部分或强烈刺激Ty 1 19个基因组缺陷rtt突变体的迁移率 保存。相比之下，两个检查点通路都不参与激活两个细胞中的Ty 1。 能够维持基因组的RTT突变体。在 rtt 101突变体，其中升高的 通过DNA损伤检查点蛋白Rad 9、Rad 24、Mec 1、Rad 53刺激转座 和Dun 1而不是Chk 1，Ty 1编码的蛋白质，而不是Ty 1 cDNA，是 检查点路径。Ty 1整合酶和逆转录酶蛋白水平，以及 逆转录酶活性，在 rtt 101突变体。我们假设DNA 在缺乏基因组完整性因子的情况下产生的损伤起触发作用， Ty 1逆转录酶活性通过S期检查点途径。

Our research concerns the mechanism and consequences of Ty element retrotransposition in the budding yeast Saccharomyces . Ty elements comprise five related families of long terminal repeat (LTR) retrotransposons that transpose via an RNA intermediate. The Ty genome contains two genes that correspond to the Gag and Pol genes of retroviruses. The retrotransposon is transcribed into a genome-length RNA, which is the template for reverse transcription by an element-encoded reverse transcriptase protein and for translation. Ty protein maturation and reverse transcription take place within Ty virus-like particles (Ty-VLPs), which appear to be essential for the transposition process. Although Ty-VLPs accumulate in the cytoplasm, a Ty preintegration complex containing Ty cDNA, the element-encoded integrase and perhaps other proteins must transit the nuclear membrane to gain access to the genome. Each Ty element class integrates nonrandomly and possesses distinctive targeting mechanisms that are influenced by the chromatin state or RNA polymerase III transcription factors. All available evidence suggests that Ty elements remain intracellular and are not infectious. Therefore, these elements and their host have evolved control mechanisms to keep transposition and element mediated genome rearrangements at a low level, and integration site preferences that reduce the possibility of causing deleterious mutations. Over the past year, we have made progress on characterizing host genes that modulate Ty1 retrotransposition. The first study involved a systematic screen of 4739 gene-deletion mutants to identify those that increase Ty1 mobility (Ty1 restriction or RTT genes). Among the 91 identified mutants, 80% encode products involved in nuclear processes such as chromatin structure and function, DNA repair and recombination, and transcription. However, bioinformatic analyses encompassing additional Ty1 and Ty3 screens indicate that 264 unique genes involved in a variety of biological processes affect Ty mobility in yeast. Further characterization of 33 of the rtt mutants identified in our screen show that Ty1 RNA levels increase in 5 mutants and the rest affect mobility posttranscriptionally. Ty1 RNA and cDNA levels remain unchanged in mutants defective in transcription elongation, including ckb2Δ and elf1Δ , suggesting Ty1 integration may be more efficient in these strains. Insertion site preference at the CAN1 locus requires Ty1 restriction genes involved in histone H2B ubiquitination by Paf complex subunit genes, as well as BRE1 and RAD6 , histone H3 acetylation by RTT109 and ASF1 , and transcription elongation by SPT5 . Our results indicate that multiple pathways restrict Ty1 mobility and histone modifications may protect coding regions from insertional mutagenesis. Since these genes are also required for efficient transcription by RNA polymerase II, additional targets for Ty1 insertion maybe uncovered by stalled transcription complexes. Ongoing work is focused on defining the Ty1 integrase targeting domain and understanding the genomic landscape available for transposition events in wild type and targeting-defective mutants. Considering the large number of genes identified in various screens that modulate Ty retrotransposition, we considered the possibility that many of the RTT genes act through a few common pathways. Support for this idea is evident from a recent study on a subset of Ty1 restriction genes, performed in collaboration with Joan Curcio's laboratory (Wadsworth Center, Albany NY). Mobility of Ty1 in budding yeast is restricted by an array of proteins that function to preserve the integrity of the genome during DNA replication and repair. However, the mechanisms involved in increasing Ty1 cDNA levels and mobility in the absence of these Rtt factors, several of which are orthologs of mammalian retroviral restriction factors, are poorly characterized. Interestingly, two S-phase checkpoint pathways, the replication stress pathway or the DNA damage pathway, partially or strongly stimulate Ty1 mobility in 19 rtt mutants with defects in genome preservation. In contrast, neither checkpoint pathway is involved in activating Ty1 in two rtt mutants that are competent for genome maintenance. In rtt101∆ mutants, in which elevated transposition is stimulated through DNA damage checkpoints proteins, Rad9, Rad24, Mec1, Rad53 and Dun1 but not Chk1, Ty1-encoded proteins, rather than Ty1 cDNA, are the direct targets of the checkpoint pathway. Levels of Ty1 integrase and reverse transcriptase proteins, as well as reverse transcriptase activity, are significantly elevated in rtt101∆ mutants. We hypothesize that DNA lesions created in the absence of genome integrity factors function as triggers that enhance Ty1 reverse transcriptase activity via S-phase checkpoint pathways.