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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：
8563519
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 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8563519
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 Sequence Rearrangement 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 public health relevance 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)可能被错误修复，导致严重的染色体重排(GCR)，如易位、大片段缺失和染色体丢失。这些GCR被认为是物种形成和人类疾病，特别是癌症的原因。在大多数人类病理中，DSB和相关的GCR的原因一直没有得到研究人员的理解。这些有害的DSB和GCR的一个潜在来源可能是RNA和同源染色体DNA序列之间形成的杂交。我的实验室使用发芽酵母作为模型系统，对杂交形成和杂交介导的GCRs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；2)一种简单的细胞学方法，用于检测单个细胞中的杂交；以及3)在YAC中的一个模型基因座，我们可以在该模型上诱导杂交形成和杂交介导的GCR。使用我们的模型基因座，我们发现变异的RNA比正常的RNA更擅长于引起杂交介导的GCR。此外，杂交体可以以反式形式形成，即在与转录本合成位置不同的位置形成。值得注意的是，这些杂交种的形成是由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，在许多在RNA生物发生的不同方面存在缺陷的突变株中，杂交和GCR的形成大大增加。我们建议在野生型和突变体中建立第一个与GCR相关的杂交种的基因组图谱。这些图谱，以及模型基因座研究，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关键的见解。我们确定了两个反杂交系统。第一种使用RNaseH，一种降解杂交种RNA的酶，第二种使用RAD51的拮抗剂Srs2。尽管对DNA修复进行了50年的密集研究，但这两种抗突变系统仍然逃脱了研究。我们建议通过遗传、生化和基因组方法来阐明这些杂交去除系统的分子功能和生物学影响。最后，杂交种介导的GCR最不为人所知的部分是杂交种向DSB的转化。我们将利用我们的模型基因座来开发检测杂交介导的双链断裂的方法，并阐明它们的形成机制。建议的研究的可行性非常高，因为我们的杂交和GCR分析的新方法，以及现有的在芽殖酵母中可用的强大方法。芽殖酵母和人类细胞在RNA和DNA代谢过程中存在着显着的保守性。新出现的研究开始显示，杂交介导的GCR也存在类似的保守性。鉴于这些先例，这项提案中的实验很可能适用于高等生物体，有可能为人类健康提供重要的见解，如癌症和出生缺陷。