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RNA:DNA hybrids and genome instability

RNA:DNA 杂交体和基因组不稳定性

基本信息

批准号：
8919922
负责人：
DOUGLAS E KOSHLAND
金额：
$ 28.43万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8919922
关键词：
Address Biochemical Biogenesis Biological Assay Biological Models Biological Process Cancer Etiology Cell Cycle Progression Cells Chromatin Chromosomal Rearrangement Chromosomes Chromosomes, Artificial, Yeast Congenital Abnormality Coupled DNA DNA Repair DNA Sequence DNA biosynthesis Disease Enzymes Excision Foundations Genes Genetic Genetic Transcription Genome Genomic Instability Genomics Health Human Human Pathology Hybrids In Vitro Individual Knowledge Laboratories Lead Length Location Malignant Neoplasms Maps Measures Mediating Messenger RNA Metabolism Methods Modeling Molecular Molecular Genetics Mutate Organism Pathway interactions Positioning Attribute Prevention Proteins RNA RNA Binding RNA Helicase RNA Processing RNA Sequence Analysis RNA annealing Reporter Research Research Personnel Ribonuclease H Role Saccharomyces cerevisiae Saccharomycetales Scientist Single-Stranded DNA Site Source System Transcript Transcription Initiation base chromosome loss ds-DNA genome-wide human disease insight mutant novel prevent repaired research study ribonuclease H1 tool

项目摘要

DESCRIPTION (provided by applicant): Project Summary: Double stranded breaks (DSBs) in DNA can be misrepaired, causing gross chromosomal rearrangements (GCRs) such as translocations, large deletions, and chromosome loss. These GCRs are thought to be causative in speciation and human disorders, especially cancer. The causes of DSBs and associated GCRs in most human pathologies have eluded researchers. One potential source of these deleterious DSBs and GCRs may be hybrids that form between RNA and homologous chromosomal DNA sequences. My laboratory has provided important new insights into hybrid formation and hybrid-mediated GCRs using budding yeast as a model system. We developed important new tools: 1) a yeast artificial chromosome (YAC) as a powerful genetic and molecular reporter of hybrid-mediated GCR; 2) a simple cytological assay to detect hybrids in individual cells; and 3) a model locus in the YAC at which we can induce hybrid formation and hybrid-mediated GCRs. Using our model locus, we showed that aberrant RNAs were much more proficient at causing hybrid mediated GCR than canonical mRNAs. In addition, hybrids can form in trans; that is, at a locus distinct from the site of transcript synthesis. Remarkably, the formation of these hybrids is mediated by the RAD51 repair pathway. We propose to exploit these findings to define the parameters in RNA and DNA (sequence, length,etc) that both promote and repress hybrid formation, DSB formation and hybrid-mediated GCR. We also showed that hybrid and GCR formation are greatly elevated in many mutants defective in diverse aspects of RNA biogenesis. We propose to generate the first genomic maps of hybrids associated with GCR in wild type and mutants. These maps, along with the model locus studies, will provide critical insights into how cis and trans factors influene the interplay between transcription, GCRs and genome instability. We identified two anti-hybrid systems. The first utilizes RNase H, an enzyme that degrades RNA in hybrids, and the second uses Srs2, an antagonist of Rad51. These two anti-mutagenic systems have escaped study despite 50 years of intensive research on DNA repair. We propose to elucidate the molecular function and biological impact of these hybrid-removal systems through genetic, biochemical and genomic methods. Finally, the least understood component of hybrid-mediated GCRs is the conversion of hybrids to DSBs. We will use our model locus to develop methods to detect hybrid-mediated DSBs and elucidate their mechanism of formation. The feasibility of the proposed studies is extremely high because of our novel assays for hybrid and GCR analysis and existing powerful approaches available in budding yeast. There is a remarkable conservation between budding yeast and human cells in the processes of RNA and DNA metabolism. Emerging studies are beginning to reveal a similar conservation for hybrid-mediated GCRs. Given these precedents, the experiments in this proposal are likely to be applicable to higher organisms, with the potential to provide important insights into human health, such as cancer and birth defects.

描述（由申请人提供）：项目摘要：DNA 中的双链断裂 (DSB) 可能会被误修复，导致染色体重排 (GCR)，例如易位、大量缺失和染色体丢失。这些 GCR 被认为是物种形成和人类疾病，尤其是癌症的原因。大多数人类病理中 DSB 和相关 GCR 的原因一直困扰着研究人员。这些有害 DSB 和 GCR 的潜在来源之一可能是 RNA 和同源染色体 DNA 序列之间形成的杂合体。我的实验室使用芽殖酵母作为模型系统，为杂交形成和杂交介导的 GCR 提供了重要的新见解。我们开发了重要的新工具：1）酵母人工染色体（YAC）作为杂交介导的 GCR 的强大遗传和分子报告基因； 2) 一种简单的细胞学检测方法来检测单个细胞中的杂交体； 3）YAC 中的一个模型位点，我们可以在其中诱导杂交形成和杂交介导的 GCR。使用我们的模型位点，我们发现异常的 RNA 比典型的 mRNA 更能熟练地引起杂交介导的 GCR。此外，杂种可以反式形成；也就是说，在不同于转录物合成位点的位点。值得注意的是，这些杂合体的形成是由 RAD51 修复途径介导的。我们建议利用这些发现来定义 RNA 和 DNA 中促进和抑制杂交形成、DSB 形成和杂交介导的 GCR 的参数（序列、长度等）。我们还表明，在 RNA 生物发生的各个方面存在缺陷的许多突变体中，杂合体和 GCR 的形成大大增加。我们建议生成第一个与野生型和突变体中 GCR 相关的杂种基因组图谱。这些图谱以及模型位点研究将为顺式和反式因子如何影响转录、GCR 和基因组不稳定性之间的相互作用提供重要见解。我们确定了两个反混合系统。第一个利用 RNase H（一种在杂交体中降解 RNA 的酶），第二个利用 Srs2（Rad51 的拮抗剂）。尽管对 DNA 修复进行了 50 年的深入研究，但这两种抗诱变系统却没有被研究。我们建议通过遗传、生化和基因组方法阐明这些杂种去除系统的分子功能和生物学影响。最后，杂种介导的 GCR 中最不为人所知的部分是杂种向 DSB 的转化。我们将使用我们的模型基因座来开发检测杂交介导的 DSB 并阐明其形成机制的方法。由于我们对杂交和 GCR 分析的新颖测定以及芽殖酵母中现有的强大方法，拟议研究的可行性非常高。芽殖酵母和人体细胞在RNA和DNA代谢过程中存在显着的保守性。新兴研究开始揭示杂交介导的 GCR 具有类似的保守性。鉴于这些先例，该提案中的实验很可能适用于高等生物，有可能为人类健康（例如癌症和出生缺陷）提供重要的见解。