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

Telomere DNA at the ends of mammalian chromosomes contains a tandemly repeated guanine-richsequence, (TTAGGG) - D2n - D2，DNA decreases with cell, telomere DNA decreases with cell, The length of telomereage, but when cells are immortalized，telomere DNA begins to elongate. The可能的联系to cell aging and cancer have attractedconsiderable interest on the telomere DNA. telomere DNA binding proteins as well as telomerase are包含telomere elongatio. TRF1 protein binds specifically with mammalian telomere DNA andnegatively regulates telomere elongation activity of telomerase. The sequence of The DNA bindingdomain (DBD) of TRF1 protein is highly homologous to that of a transcription factorMyb.In the present study，我们发现，即使是Myb-like DBD of human and mouse TRF1 proteins had the ability to bindwith the telomere DNA specifically and strongly as in the case of the full-length TRF1. A 16-bqtelomere DNA sequence was the minimal length ne更多需求for the specific binding with the human and mouse TRF1 DBDs. the peptides of theDBDs and the oligonucleotides of the target telomere DNA sequence were synthesized by peptide andDNA synthesizer, respectively，and purified with a reverse phase HPLC. We are searching for the best condition to crystallize thecomplex between the peptides of the DBDs and the oligonucleotides. In addition，我们established an E. coli expression system of the gene for the human and mouse full-lengthTRF1 and the purification scheme of the expressed proteins. Since the yield of the proteinsexpressed in E. coli not good enough for crystallization我们正在努力创造改变minor codons around the initiation codon into majorcodons in the E. coli expression plasmid or by using an in vitro cell-free protein expressionsystem. The crystallization of The complex between The human and mouse full-length TRF1 expressed in The crystallization of The complexE. coli and the telomere DNA oligonucleotides is underway to determine the three-dimensionalstruct of the complex. Less

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