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重复序列，在人类基因组中扩增，导致17种遗传性人类疾病，包括亨廷顿氏病、肌强直性营养不良症和脊髓小脑共济失调。这些序列强大的发夹形成潜力可能是扩大tnr时基因组不稳定的基础。扩大的tnr抑制DNA间隙的修复，干扰DNA复制，并导致染色体断裂（染色体脆性）。我们的研究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。我们建议以酿酒酵母为模型系统，描述参与DNA修复、DNA损伤检查点和染色质修饰的蛋白质在防止CAG/CTG重复脆性和扩增中的作用。研究人员将利用染色质免疫沉淀和荧光技术研究强发夹形成序列对自然发生损伤的修复反应，以确定扩展tnr的修复蛋白募集的身份和时间。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2d凝胶确定DNA损伤检查点在防止长重复序列复制叉停滞中的作用。将通过确定扩展TNR的组蛋白修饰状态和间距来研究TNR稳定性和染色质结构之间的新联系。最后，我们提出了一种遗传筛选方法来鉴定在防止CAG/CTG束染色体脆性中重要的其他蛋白质和途径。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以及预防亨廷顿病非分裂脑细胞的病理性体细胞扩增都很重要。由于细胞死亡是几种TNR疾病进展的主要因素，确定扩展重复序列损伤对细胞健康的影响可能导致减缓疾病发展的策略。人类基因组包含许多在复制和修复过程中存在潜在问题的直接重复和反向重复以及其他类型的序列，因此了解已经进化到应对这些问题的细胞机制应该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.