Regulation of Targeted Gene Correction

靶向基因校正调控

• 批准号: 6514843
• 负责人: Eric B. Kmiec
• 金额: $ 25.69万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6514843
• 关键词: DNA binding protein; DNA repair; bacterial proteins; cell free system; crosslink; drug resistance; frameshift mutation; fungal genetics; fungal proteins; fusion gene; oligonucleotides; point mutation; psoralens; synapses; transposon /insertion element; yeasts

Chimeric DNAJRNA double hairpins are synthetic oligonucleotides that direct targeted nucleotide substitutions in bacteria, yeast, plants and animals. We have postulated that cellular recombinases catalyze joint molecule formation between these oligonucleotides and homologous dsDNA. According to our model, resolution of these joints can be accompanied by point mutation of the target DNA when the oligonucleotide is mismatched within the intermediate joint. Since chimeric hairpins are effective gene repair agents in yeast, we propose to validate our model in this microorganism. Experiments will be carried out with whole cells, cell-free extracts, and purified protein with the goal of delineating the mechanism of joint molecule formation and gene substitution or frameshift mutations in yeast will be monitored by targeting episomal or chromosomal genes that express a fluorescent signal or an antibiotic resistance marker. Targeting of the episomal target in different knock out strains will drive the discovery of formation between chimeric hairpins and dsDNA will be optimized using gene products from the RAD52 epistasis group either alone or in combination with other related enzymes. Nucleoprotein intermediates formed during strand exchange will be probed to elucidate how a chimeric DNAIRNA backbone enhances recombination. Performed double D-loop joints that contain a specific mismatch or unpaired base will be used as substrates to study targeted nucleotides exchange. The mechanism which emerges from this study should provide guidance in the use of chimeric hairpin oligonucleotides in other systems including plant, animal and human cells.

嵌合DNAJRNA双发夹是合成寡核苷酸 直接靶向细菌，酵母，植物和 动物。我们假设细胞重组酶催化关节分子 这些寡核苷酸与同源dsDNA之间的形成。根据我们的说法 模型，这些关节的分辨率可以伴随着点突变 靶DNA当寡核苷酸在中间位置不匹配时 联合的。由于嵌合发夹是酵母中有效的基因修复剂，我们 建议在这种微生物中验证我们的模型。实验将是 用全细胞，无细胞提取物和纯化的蛋白 描述关节分子形成和基因机制的目标 酵母中的替代或移码突变将通过靶向监测 表达荧光信号或染色体基因或染色体基因 抗生素抗性标记。靶向偶发目标不同 敲除菌株将推动嵌合之间的形成 发夹和DSDNA将使用Rad52的基因产物进行优化 上静脉体组单独或与其他相关酶结合使用。 在链交换过程中形成的核蛋白中间体将被探测到 阐明嵌合型执层骨架如何增强重组。执行 包含特定不匹配或未配对基座的双D环关节将是 用作研究靶向核苷酸交换的底物。机制 从这项研究中出现的应该为使用嵌合发夹提供指导 其他系统中的寡核苷酸，包括植物，动物和人类细胞。