Higher-order structure and triplex DNA recognition mechanism of triplex DNA binding protein

三链体DNA结合蛋白的高阶结构及三链体DNA识别机制

• 批准号: 13670133
• 负责人: TORIGOE Hidetaka
• 金额: $ 2.3万
• 依托单位: Tokyo University of Science (2002)The Institute of Physical and Chemical Research (2001)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670133/
• 关键词: Triplex DNA; DNA binding protein; Higher-order structure; DNA Recognition Mechanism; 結合定数; 分子認識

A triplex is usually formed when a homopyrimidine or homopurine single strand binds to the major groove of homopurine-homopyrimidine stretch in duplex DNA via Hoogsteen hydrogen bonding. The homopurine-homopyrimidine stretch is widely distributed in eukaryotic genome and often located in the region controlling gene expression and the hotspot of genetic recombination. Thus, the triplex is considered to be involved in the control of gene expression and genetic recombination. In the present study, we have analyzed the properties of a recently isolated triplex DNA-binding protein, STM1. First, we have constructed the expression system of STM1 under the control of T7 promoter in E. coli. We have also expressed STM1 in a fusion protein with glutathione S-transferase under the control of tac promoter in E. coli. The amount of the expressed protein was larger in the latter system. We have achieved the purification of the expressed protein. Next, we have examined the binding affinity of STM1 wi … More th a series of the following DNA structures, 1) triplex DNA between a homopurine single strand and a homopurine-homopyrimidine duplex DNA (Pur triplex), 2) triplex DNA between a homopyriminde single strand and a homopurine-homopyrimidine duplex DNA (Pry triplex), 3) duplex DNA between G-rich and C-rich single strands (Pur duplex), 4) duplex DNA between A-rich and T-rich single strands (Pry duplex), 5) G-rich single strand, and 6) tetraplex DNA with folded G-rich single strand (G-tetramer). STM1 showed the highest binding affinity with the Pur triplex among these DNA structures. The binding affinity of STM1 with each of the Pur triplex and the Pur duplex was a few ten times smaller than that with the Pur triplex. STM1 did not bind to the Pyr duplex. The binding constant of STM1 with the G-rich single strand was a few thousand times smaller than that with the Pur triplex. On the other hand, STM1 showed the relatively larger binding affinity with the G-tetramer, although the binding constant with the G-tetramer was several times smaller than that with the Pur triplex. A part of telomere region at the end of linear chromosome is composed of G-rich single strand and has the potential to form the tetraplex DNA. Combining the present result with the potential of the telomere region to form the tetraplex DNA, STM1 may be involved in the maintenance of the telomere structure. In addition, we have also found that the C-terminal region of STM1 may directly interact with the nucleic acids Less

当高嘌呤或高嘌呤单链通过Hoogsteen氢键结合到双链DNA中高嘌呤-高嘧啶伸展的主要凹槽上时，通常形成三链。高嘌呤-高嘧啶伸展广泛存在于真核生物基因组中，常位于基因表达调控区域和基因重组的热点区域。因此，三联体被认为参与了基因表达和基因重组的调控。在本研究中，我们分析了最近分离的三链DNA结合蛋白STM1的性质。首先，我们构建了T7启动子调控下的STM1基因在大肠杆菌中的表达系统。我们还在Tac启动子控制下的谷胱甘肽S转移酶融合蛋白中进行了表达。在后一种系统中，表达的蛋白质数量较多。我们已经实现了表达蛋白的纯化。接下来，我们研究了STM1wi…的结合亲和力更多的是以下一系列DNA结构，1)高嘌呤单链和高嘌呤-高嘧啶双链DNA之间的三链DNA(PUR三链)，2)高吡啶单链和高嘌呤-高嘧啶双链DNA之间的三链DNA(撬三链)，3)富含G和C的单链之间的双链DNA(PUR双链)，4)富A和富T单链之间的双链DNA(撬双链)，5)富含G的单链和折叠的四链DNA(G-四聚体)。在这些DNA结构中，STM1与pur三联体的结合亲和力最高。STM1与PUR三联体和PUR双链的结合亲和力比与PUR三联体的结合亲和力小几十倍。STM1不与PYR双链结合。STM1与富含G的单链的结合常数比与PUR三链的结合常数小几千倍。另一方面，STM1与G-四聚体的结合亲和力相对较大，但与G-四聚体的结合常数比与PUR三聚体的结合常数小好几倍。线形染色体末端的部分端粒区由富含G的单链组成，具有形成四链DNA的潜力。结合目前的结果和端粒区形成四链DNA的潜力，STM1可能参与端粒结构的维持。此外，我们还发现，STM1的C-末端区域可能与核酸直接作用较少

项目成果

Torigoe, H., Hari, Y., Obika, S., Imanishi, T.: "Triplex Formation Involving 2'-4'-BNA with 2-Pyridone Base Analogue : Efficient and Selective Recognition of C : G Interruption"Nucleic Acids Res.. 29・sl. 281-282 (2001)

Torigoe, H.、Hari, Y.、Obika, S.、Imanishi, T.：“涉及 2-4-BNA 与 2-吡啶酮碱类似物的三链体形成：有效且选择性地识别 C : G 中断”核酸决议.. 29・sl. 281-282 (2001)

Torigoe, H.: "Thermodynamic and Kinetic Effects of N3'-P5' Phosphoramidate Modification on Pyrimidine Motif Triplex DNA Formation"Biochemistry. 40. 1063-1069 (2001)

Torigoe, H.：“N3-P5 氨基磷酸酯修饰对嘧啶基序三链体 DNA 形成的热力学和动力学影响”生物化学。

Torigoe, H. Sato, S., Yamashita, K., Obika, S., Imanishi, T., and Takenaka, S.: "Binding of Threading Intercalator to Nucleic Acids: Thermodynamic Analyses"Nucleic Acids Res.. 30, s2. *****-***** (2002)

Torigoe, H. Sato, S.、Yamashita, K.、Obika, S.、Imanishi, T. 和 Takenaka, S.：“螺纹嵌入剂与核酸的结合：热力学分析”核酸研究.. 30，s2

Torigoe, H., Obika, S., Imanishi, T.: "Promotion of Triplex Formation by 2'-O,4'-C-Methylene Bridged Nucleic Acid (2',4'-BNA) Modification : Thermodynamic and Kinetic Studies"Nucleosides, Nucleotides & Nucleic Acids. 20. 1235-1238 (2001)

Torigoe, H.、Obika, S.、Imanishi, T.：“通过 2-O,4-C-亚甲基桥核酸 (2,4-BNA) 修饰促进三链体形成：热力学和动力学研究

Sasaki, S., Shibata, T., Torigor, H. Shibata, Y., and Maeda, M.: "Novel Class of DNA Binding Motifs Based on Bistetrahydrofuran and Bisfuran Skeleton with Long Alkyl Chains"Nucleosides, Nucleotides & Nucl*** Acids. 20. 551-558 (2001)

Sasaki, S.、Shibata, T.、Torigor, H. Shibata, Y. 和 Maeda, M.：“基于具有长烷基链的双四氢呋喃和双呋喃骨架的新型 DNA 结合基序”核苷、核苷酸