Fragility and Instability at Hairpin-Forming Trinucleotide Repeats in Yeast

描述（由申请人提供）：三核苷酸重复序列（TNR），如CAG/CTG重复，在人类基因组中扩增，引起17种遗传性人类疾病，包括亨廷顿病、强直性肌营养不良和脊髓小脑共济失调。这些序列的强发夹形成潜力可能是扩增TNR时基因组不稳定性的基础。扩增的TNR抑制DNA缺口的修复，干扰DNA复制，并导致染色体断裂（染色体脆性）。我们研究的目标是阐明通常促进TNR复制和修复的细胞机制，并确定这些系统的失败如何导致重复扩增和染色体断裂。我们建议的特点蛋白质参与DNA修复，DNA损伤检查点，染色质修饰，防止CAG/CTG重复脆性和扩张的作用，使用酿酒酵母作为模型系统。将通过使用染色质免疫沉淀和荧光技术来研究对强发夹形成序列处天然发生的损伤的修复反应，以确定修复蛋白招募到扩增的TNR的身份和时间。为了确定TNR激活DNA损伤检查点的细胞后果，将监测含有长CAG/CTG重复序列的单细胞的分裂，并通过2-D凝胶确定DNA损伤检查点在防止复制叉在长重复序列处停滞中的作用。TNR稳定性和染色质结构之间的新联系将通过确定组蛋白修饰的状态和扩展TNR的间距来研究。最后，我们提出了一个遗传筛选，以确定其他蛋白质和途径，重要的是防止染色体脆性在CAG/CTG道。了解CAG/CTG序列的修复如何发生对于解释重复扩增疾病如何发生和预防亨廷顿病中非分裂脑细胞的病理性体细胞扩增都是重要的。由于细胞死亡是几种TNR疾病进展的主要因素，因此确定细胞健康的扩展重复序列的损伤后果可能会导致减缓疾病发展的策略。人类基因组包含许多正向和反向重复序列以及其他类型的序列，这些序列在复制和修复过程中存在潜在问题，因此了解为科普这些问题而进化的细胞机制应该会对基因组稳定性产生重要的见解。

DESCRIPTION (provided by applicant): Trinucleotide repeat sequences (TNRs), such as CAG/CTG repeats, expand in the human genome to cause 17 inherited human diseases, including Huntington's disease, myotonic dystrophy, and spinocerebellar ataxias. The strong hairpin-forming potential of these sequences is the likely basis for genome instability at expanded TNRs. Expanded TNRs inhibit repair of DNA gaps, interfere with DNA replication, and cause chromosomes to break (chromosome fragility). The goal of our research is to elucidate the cellular mechanisms that normally facilitate replication and repair of TNRs and determine how failure of these systems can lead to repeat expansion and chromosome breakage. We propose to characterize the role of proteins involved in DNA repair, the DNA damage checkpoint, and chromatin modification in preventing CAG/CTG repeat fragility and expansion, using Saccharomyces cerevisiae as a model system. The repair response to naturally occurring damage at strong hairpin-forming sequences will be investigated by using chromatin immunoprecipitation and fluorescence techniques to determine the identity and timing of repair protein recruitment to expanded TNRs. To establish the cellular consequences of DNA damage checkpoint activation by TNRs, division of single cells containing long CAG/CTG repeats will be monitored, and the role of the DNA damage checkpoint in preventing replication fork stalling at long repeats ascertained by 2-D gels. A novel link between TNR stability and chromatin structure will be investigated by determining the state of histone modifications and spacing at expanded TNRs. Lastly, we propose a genetic screen to identify additional proteins and pathways important in preventing chromosome fragility at CAG/CTG tracts. An understanding of how repair of CAG/CTG sequences occurs is important both to explain how repeat expansion diseases occur and to prevent pathological somatic expansions in non-dividing brain cells in Huntington's disease. Since cell death is a major factor in progression of several TNR diseases, determining the consequences of damage at expanded repeats for cell health could lead to strategies to slow disease development. The human genome contains many direct and inverted repeats and other types of sequences that present potential problems during replication and repair, so understanding the cellular mechanisms that have evolved to cope with these problems should yield important insights into genome stability.