The study of inter-molecular interaction between polyQ strands that promote polyQ-containing proteins co-aggregation and the role of Bem2p plays in the DNA stability of repetitive sequence that encodes polyQ

• Main Authors: Tzu-Han Wang, 王慈涵
• Other Authors: Tzu-Hao Cheng
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/21984963689018542246

碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 97 === 1.Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder and is caused by the expansion of CAG repeats, encoding polyglutamines (polyQ), within exon 1 of the huntingtin (htt) gene. The length of polyQ track is a main determinant for the onset of HD and the disease occurs only when the critical length of ~36 glutamines is exceeded. A peculiar feature of mutant htt proteins with abnormal length of polyQ is that they spontaneously self-assemble into aggregates, a pathological hallmark of this disorder found in the brain of affected individuals. Several studies have demonstrated that proteins with normal length of polyQ track can also interact with and recruit into these aggregates, which are initiated by polyQ-expanded proteins. In order to further investigate this inter-molecular interaction between heterogeneous polyQ strands and to find a potential dominant negative mutant that can interfere with the formation of polyQ aggregates in cells, we have established a phenotypic assay system that can reveal the presence of co-aggregation and costructed a library of amino acid substitution within the polyQ track for examining their effect on polyQ aggregation. After three rounds of screening, we find that co-expression of truncated polyQ peptides can partially reduce the aggregation levels of 97Q protein. However, this effect may result from decreased 97Q expression. 2.The BEM2 gene is required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. In the carboxyl-terminus, Bem2p contains a GAP domain, which is commoly found in GTPase activating proteins (GAPs) that regulate rho-GTPase activity. In yeast, genes encoding GTPases (CDC42, RHO1, RHO2, RHO3, and RHO4) have been proposed to regulate cellular morphogenesis such as actin cytoskeleton and bud emergence. Using the Snyder’s knock out library, we find that bem2△ mutations confer DNA repetitive sequence instability and enhance contraction of CAA/CAG repeats through homologous recombination, suggesting Bem2p has a role in regulating DNA stability. We also demonstrate the DNA instability is attributed to the defective function of Bem2p in morphogenesis checkpoint. Furthermore, swe1, a△ morphogenesis checkpoint deficient cell, shows an elevated DNA contraction rate. Thus, our study reveals a novel link between morphogenesis checkpoint and repetitive sequence stability in yeast cells.