The contribution of fragile site structure to genome instability in humanized yea

描述（申请人提供）：染色体脆性位点具有多种结构，但脆性位点的结构与其稳定性之间没有明确的相关性。因此，很难为脆性位点的不稳定性设计一个简单的解释，这种知识上的差距使我们既无法理解这些序列改变染色体结构的机制，也无法开发保护基因组完整性的治疗干预措施。本申请的目的是使用酵母作为模型系统，以了解反转录转座子DNA的排列如何产生脆性位点并改变DNA基因座进行重排的倾向。酵母染色体III上的一对反向反转录转座子遭受双链断裂和染色体易位的发生率增加，这表明串联或反向方向存在的重复序列起脆性位点的作用。这项研究的核心假设是，重复DNA的含量和组织将是决定遗传位点不稳定性频率的关键因素。中心假设将通过使用一系列等基因的反转录转座子过量（RO）菌株进行测试，这些菌株含有大量分散在整个基因组中的反转录转座子，包括许多对作为潜在脆弱位点的反转录转座子;这些菌株是一个独特的遗传系统，使研究能够在同一基因组中的多个脆弱位点。将通过追求以下具体目标来研究脆弱位点结构对基因组不稳定性的贡献：（1）鉴定RO菌株中复制应激后最容易不稳定的基因组基因座，以及（2）确定实验室进化后预测的脆弱位点的相对稳定性。这一创新办法预计将产生以下成果。第一，将为人类脆弱地点开发一个更可行的模型。该RO模型系统可以代表人类脆性位点序列和结构的多样性，从而阐明为什么人类脆性位点在其稳定性方面有显着差异。第二，酵母fagile网站的进化稳定性将被确定，从而阐明为什么脆弱的网站在进化过程中保持，以及它们是否与保守的染色体断裂点。拟议研究的结果将是对酵母中DNA结构和基因组稳定性之间关系的更透彻理解，这是理解染色体脆性机制的先决条件。 公共卫生相关性：脆性位点是易于不稳定的染色体区域，可能由DNA断裂发生率增加引起的染色体重排代表了癌症进展中的一些最早变化。这项研究将采用一种新的“人源化”酵母模型系统，包含不同结构的脆性位点。拟议的研究与公共卫生有关，因为它将使我们了解为什么脆弱位点的稳定性差异很大，从而使我们能够监测或靶向特定的脆弱位点，以防止基因组不稳定。

DESCRIPTION (provided by applicant): Chromosome fragile sites have diverse structures, yet there is no clear correlation between the structure of a fragile site and its stability. It is therefore difficult to devise a simple explanation for fragile site instability, and this gap in knowledge prevents us both from understanding the mechanism by which these sequences alter chromosome structure and also from developing therapeutic interventions to preserve genome integrity. The objective of this application is to use yeast as a model system to understand how the arrangement of retrotransposon DNA can create fragile sites and alter the propensity of a DNA locus to undergo rearrangement. A pair of inverted retrotransposons on yeast chromosome III suffers an increased incidence of double strand breaks and chromosome translocations, suggesting that repetitive sequences that are present in tandem or inverted orientation function as fragile sites. The central hypothesis for the proposed research is that repetitive DNA content and organization will be a crucial factor in determining the frequency with which genetic loci display instability. The central hypothesis will be tested by using an isogenic series of retrotransposon overdose (RO) strains that contain an elevated number of retrotransposons dispersed throughout the genome, including numerous pairs of retrotransposons that are potential fragile sites; these strains are a unique genetic system that enable the study multiple fragile sites within the same genome. The contribution of fragile site structure to genome instability will be investigated by pursuing the following specific aims: (1) identifying genomic loci that are most prone to instability following replication stress in RO strains, and (2) determining the relative stability of predicted fragile sites following lab evoluton. This innovative approach is expected to produce the following outcomes. First, a more viable model for human fragile sites will be developed. This RO model system can represent the diversity in sequence and structure of human fragile sites, thereby elucidating why human fragile sites differ dramatically in their stability. Second, the evolutionary stability of yeast fagile sites will be determined, thereby elucidating why fragile sites are maintained during evolution and whether they correlate with conserved chromosome breakpoints. The outcome of the proposed studies will be a more thorough understanding of the relationship between DNA structure and genome stability in yeast, a prerequisite to understanding the mechanism of chromosome fragility. PUBLIC HEALTH RELEVANCE: Fragile sites are chromosome regions that are prone to instability, and chromosome rearrangements that may result from this increased incidence of DNA breakage represent some of the earliest changes in cancer progression. This study will employ a novel "humanized" yeast model system that contains fragile sites with diverse structures. The proposed research is relevant to public health because it will allow us to understand why fragile sites differ dramatically in their stability, thereby allowing us to monitoror target specific fragile sites to prevent genome instability.