Ty Element Retrotransposition in Saccharomyces cerevisiae

酿酒酵母中的 Ty 元件逆转录转座

• 批准号: 7733009
• 负责人: David J. Garfinkel
• 金额: $ 129.68万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733009
• 关键词: AIDS/HIV problem; Acetylation; Affect; Basic Science; Bioinformatics; Biological Preservation; Biological Process; Cancerous; Cell physiology; Cells; Chromatin; Chromatin Structure; Class; Classification; Code; Collaborations; Complementary DNA; Complex; Coupled; Cytoplasm; DNA Damage; DNA Repair; DNA Sequence Rearrangement; DNA biosynthesis; DNA damage checkpoint; DNA lesion; Defect; Disease; Elements; Event; Family; Gagging; Gene Deletion; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genome; Genomic Instability; Genomics; Goals; HIV; Histone H2B; Histone H3; Histones; Human; Human Genome; Insertional Mutagenesis; Integrase; Laboratories; Learning; Length; Long Terminal Repeats; Maintenance; Malignant Neoplasms; Mediating; Mobile Genetic Elements; Modeling; Modification; Mutation; Nuclear; Nuclear Envelope; Numbers; Orthologous Gene; Pathway interactions; Phase; Predisposition; Process; Proteins; RNA; RNA Interference Pathway; RNA Polymerase II; RNA Polymerase III Transcription Pathway; RNA-Directed DNA Polymerase; Research; Research Project Grants; Rest; Retroelements; Retrotransposition; Retrotransposon; Retroviridae; Reverse Transcription; Role; Saccharomyces; Saccharomyces cerevisiae; Saccharomycetales; Short Interspersed Nucleotide Elements; Site; Stress; Transcription Elongation; Translations; Ubiquitination; Virus-like particle; Work; Yeasts; carcinogenesis; functional genomics; genome sequencing; interest; mutant; particle; pol genes; preference; protein function; rad9 protein; repaired; tool; trafficking; transcription factor

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.

我们的研究是关于Ty元件在芽殖酵母中逆转录转座的机制和后果。TY元件由五个相关的长末端重复序列(LTR)反转录转座子家族组成，它们通过RNA中间体转座。Ty基因组包含两个基因，分别对应于逆转录病毒的Gag和Pol基因。反转录转座子被转录成基因组长度的RNA，该RNA是由元件编码的逆转录酶蛋白进行逆转录和翻译的模板。TY蛋白的成熟和逆转录发生在TY病毒样颗粒(TY-VLP)内，这似乎是转座过程中必不可少的。尽管Ty-VLPs积累在细胞质中，但Ty预整合复合体必须通过核膜才能进入基因组，该复合体包含Ty cDNA、元件编码的整合酶以及其他蛋白质。每一类Ty元件都是非随机整合的，并具有受染色质状态或RNA聚合酶III转录因子影响的独特靶向机制。所有可获得的证据表明，TY分子仍然存在于细胞内，不具传染性。因此，这些元件和它们的宿主进化出了控制机制，将转座和元件介导的基因组重排保持在较低水平，并进化出整合位点偏好，从而降低了导致有害突变的可能性。在过去的一年里，我们在确定调节Ty1逆转录转座的宿主基因方面取得了进展。第一项研究涉及对4739个基因缺失突变的系统筛选，以确定那些增加Ty1迁移率的突变(Ty1限制或RTT基因)。在91个已鉴定的突变体中，80%编码与染色质结构和功能、DNA修复和重组以及转录等核过程有关的产物。然而，包括额外的Ty1和Ty3筛查的生物信息学分析表明，参与各种生物学过程的264个独特基因影响酵母中Ty的迁移率。对我们筛选中发现的33个RTT突变体的进一步鉴定表明，5个突变体的Ty1 RNA水平上升，其余的影响转录后的迁移率。在转录延伸缺陷的突变株中，Ty1的RNA和cDNA水平保持不变，包括CKB2和Elf1；，这表明Ty1整合在这些菌株中可能更有效。CAN1位点的插入位点偏好需要组蛋白H_2B由PAF复合亚单位基因泛素化所涉及的Ty1限制基因，以及由RTT109和ASF1导致的组蛋白H3乙酰化以及由SPT5引起的转录延伸。我们的结果表明，多条途径限制了Ty1的移动，组蛋白修饰可能保护编码区免受插入突变。由于这些基因也是RNA聚合酶II有效转录所必需的，因此，停滞的转录复合体可能会发现Ty1插入的其他靶点。正在进行的工作集中在定义Ty1整合酶靶向结构域和了解野生型和靶向缺陷突变体中可用于转座事件的基因组图景。考虑到在各种筛选中发现了大量调节Ty逆转录转座的基因，我们考虑了许多RTT基因通过几个常见途径发挥作用的可能性。最近与Joan Curcio的实验室(纽约州奥尔巴尼沃兹沃斯中心)合作进行的一项关于Ty1限制基因子集的研究明显支持这一观点。在萌芽酵母中，Ty1的移动性受到一系列蛋白质的限制，这些蛋白质在DNA复制和修复过程中起到保护基因组完整性的作用。然而，在缺乏这些RTT因子(其中几个是哺乳动物逆转录病毒限制因子的直系物)的情况下，增加Ty1基因水平和迁移率的机制尚不清楚。有趣的是，两个S阶段的检查点途径，复制应激途径或DNA损伤途径，部分或强烈地刺激了19个基因组保存缺陷的rtt突变体的Ty1迁移率。相反，在两个有能力进行基因组维护的RTT突变体中，这两个检查点途径都不参与激活Ty1。在rtt101&#8710；突变体中，通过DNA损伤检查点蛋白Rad9、Rad24、Mec1、Rad53和Dun1来刺激提升的转座，而不是Chk1、Ty1编码的蛋白而不是Ty1cDNA是检查点途径的直接靶标。在rtt101和#8710；突变体中，Ty1整合酶和逆转录酶蛋白水平以及逆转录酶活性显著升高。我们假设，在缺乏基因组完整性因素的情况下产生的DNA损伤作为触发器，通过S阶段的检查点途径增强Ty1逆转录酶的活性。

项目成果

The Rad27 (Fen-1) nuclease inhibits Ty1 mobility in Saccharomyces cerevisiae.

Rad27 (Fen-1) 核酸酶抑制酿酒酵母中的 Ty1 迁移性。

• DOI: 10.1093/genetics/163.1.55
• 发表时间: 2003
• 期刊: Genetics
• 影响因子: 3.3
• 作者: Sundararajan,Anuradha;Lee,Bum-Soo;Garfinkel,DavidJ
• 通讯作者: Garfinkel,DavidJ

Sensitive phenotypic detection of minor drug-resistant human immunodeficiency virus type 1 reverse transcriptase variants.

轻微耐药人类免疫缺陷病毒 1 型逆转录酶变体的灵敏表型检测。

• DOI: 10.1128/jcm.43.11.5696-5704.2005
• 发表时间: 2005
• 期刊: Journal of clinical microbiology
• 影响因子: 9.4
• 作者: Nissley,DwightV;Halvas,EliasK;Hoppman,NicoleL;Garfinkel,DavidJ;Mellors,JohnW;Strathern,JeffreyN
• 通讯作者: Strathern,JeffreyN

Survival strategies for transposons and genomes.

转座子和基因组的生存策略。

• DOI: 10.1186/gb-2003-4-4-313
• 发表时间: 2003
• 期刊: Genome biology
• 影响因子: 12.3
• 作者: Martin,SandraL;Garfinkel,DavidJ
• 通讯作者: Garfinkel,DavidJ

Correct integration of model substrates by Ty1 integrase.

Ty1 整合酶正确整合模型底物。

• DOI: 10.1128/jvi.74.24.11522-11530.2000
• 发表时间: 2000
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Moore,SP;Garfinkel,DJ
• 通讯作者: Garfinkel,DJ