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Higher-order structure and telomere DNA recognition mechanism of telomere DNA binding protein TRF1

端粒DNA结合蛋白TRF1的高阶结构及端粒DNA识别机制

基本信息

批准号：
10680593
负责人：
TORIGOE Hidetaka
金额：
$ 1.86万
依托单位：
The Institute of Physical and Chemical Research (RIKEN)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680593/
关键词：
Telomere DNA DNA binding protein Higher order structure DNA Recognition Mechanism

项目摘要

Telomere DNA at the ends of mammalian chromosomes contains a tandemly repeated guanine-rich sequence, (TTAGGG)ィイD2nィエD2, and is essential for maintaining chromosal stability. The length of telomere DNA decreases with cell age, but when cells are immortalized, telomere DNA begins to elongate. The possible relations to cell aging and cancer have attracted considerable interest on the telomere DNA. Telomere DNA binding proteins as well as telomerase are involved in telomere elongatio. TRF1 protein binds specifically with mammalian telomere DNA and negatively regulates telomere elongation activity of telomerase. The sequence of the DNA binding domain (DBD) of TRF1 protein is highly homologous to that of a transcription factor, Myb.In the present study, we have found that even the Myb-like DBD of human and mouse TRF1 proteins had the ability to bind with the telomere DNA specifically and strongly as in the case of the full-length TRF1. A 16-bq telomere DNA sequence was the minimal length ne … More cessary for the specific binding with the human and mouse TRF1 DBDs. The peptides of the DBDs and the oligonucleotides of the target telomere DNA sequence were synthesized by peptide and DNA synthesizer, respectively, and purified with a reverse phase HPLC. We are searching for the best condition to crystallize the complex between the peptides of the DBDs and the oligonucleotides. In addition, we have established an E. coli expression system of the gene for the human and mouse full-length TRF1 and the purification scheme of the expressed proteins. Since the yield of the proteins expressed in E. coli was not good enough for crystallization, we are trying to increase the yield by changing minor codons around the initiation codon into major codons in the E. coli expression plasmid or by using an in vitro cell-free protein expression system. The crystallization of the complex between the human and mouse full-length TRF1 expressed in E. coli and the telomere DNA oligonucleotides is underway to determine the three-dimensional structure of the complex. Less

哺乳动物染色体末端的端粒DNA含有一个串联重复的富含鸟嘌呤的序列（TTAGGG）端粒D2 n端粒D2，它是维持染色体稳定性所必需的。端粒DNA的长度随着细胞年龄的增长而减少，但当细胞永生化时，端粒DNA开始伸长。端粒DNA与细胞衰老和癌症的可能关系引起了人们对端粒DNA的极大兴趣。端粒DNA结合蛋白和端粒酶都参与端粒的延长。TRF 1蛋白与哺乳动物端粒DNA特异性结合，负性调节端粒酶的端粒延伸活性。TRF 1蛋白的DNA结合结构域（DBD）序列与转录因子Myb的序列高度同源。在本研究中，我们发现，即使是人和小鼠TRF 1蛋白的Myb样DBD也具有与端粒DNA特异性和强烈结合的能力，就像全长TRF 1的情况一样。一个16-bq端粒DNA序列是最小长度的， ...更多信息与人和小鼠TRF 1 DBD特异性结合的条件。分别用肽合成仪和DNA合成仪合成DBD的肽和靶端粒DNA序列的寡核苷酸，并用反相HPLC纯化。我们正在寻找最佳的条件来结晶DBD肽和寡核苷酸之间的复合物。此外，我们还建立了一个E.人和小鼠全长TRF 1基因的大肠杆菌表达系统和表达蛋白的纯化方案。由于E.大肠杆菌中的起始密码子不适合结晶，我们试图通过将起始密码子周围的次要密码子改变为大肠杆菌中的主要密码子来提高产量。coli表达质粒或使用体外无细胞蛋白表达系统。在大肠杆菌中表达的人和小鼠全长TRF 1之间的复合物的结晶。大肠杆菌和端粒DNA的寡核苷酸正在进行中，以确定复合物的三维结构。少