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

当高嘧啶或高嘌呤单链通过Hoogsteen氢键与双链体DNA中高嘌呤-高嘧啶段的大沟结合时，通常形成三链体。高嘌呤-高嘧啶片段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热点区域。因此，三链体被认为参与基因表达和遗传重组的控制。在本研究中，我们分析了最近分离的三链DNA结合蛋白，STM 1的属性。首先，我们构建了T7启动子控制下的STM 1在大肠杆菌中的表达系统。杆菌我们还在大肠杆菌中表达了在tac启动子控制下的STM 1与谷胱甘肽S-转移酶的融合蛋白。杆菌在后一种系统中表达的蛋白量较大。我们实现了表达蛋白的纯化。接下来，我们已经检查了STM 1的结合亲和力， ...更多信息 它们是一系列DNA结构：1）高嘌呤单链DNA和高嘌呤-高嘧啶双链DNA之间的三链DNA（Pur三链体），2）高嘧啶单链和高嘌呤-高嘧啶双链体DNA之间的三链体DNA（Pry三链体），3）富含G和富含C的单链之间的双链体DNA（Pur双链体），4）富含A和富含T的单链之间的双链体DNA（Pry双链体），5）富含G的单链，和6）具有折叠的富含G的单链的四链体DNA（G-四聚体）。在这些DNA结构中，STM 1与Pur三链体的结合亲和力最高。STM 1与Pur三链体和Pur双链体的结合亲和力比与Pur三链体的结合亲和力小几十倍。STM 1不与Pyr双链体结合。STM 1与富含G的单链的结合常数比与Pur三链体的结合常数小几千倍。另一方面，STM 1与G-四聚体显示出相对较大的结合亲和力，尽管与G-四聚体的结合常数比与Pur三链体的结合常数小几倍。位于线状染色体末端的端粒区部分由富含G的单链组成，具有形成四链体DNA的潜力。结合端粒区域形成四链体DNA的潜力，STM 1可能参与端粒结构的维持。此外，我们还发现STM 1的C-末端区域可能直接与核酸相互作用。

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