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

当同嘧啶或同嘌呤单链通过Hoogsteen氢键与双链DNA中同嘌呤-同嘧啶拉伸的主槽结合时，通常形成三联体。同嘌呤-同嘧啶链广泛分布于真核生物基因组中，常位于基因表达控制区域和基因重组热点区域。因此，三重体被认为参与基因表达和基因重组的控制。在本研究中，我们分析了最近分离的三重dna结合蛋白STM1的特性。首先，我们在大肠杆菌中构建了T7启动子控制下STM1的表达体系。我们还在大肠杆菌tac启动子的控制下，将STM1与谷胱甘肽s -转移酶融合表达。后一种系统表达的蛋白量较大。我们已经完成了表达蛋白的纯化。接下来，我们研究了STM1与以下一系列DNA结构的结合亲和性，1)同嘌呤单链与同嘌呤-同嘧啶双链之间的三联体DNA （Pur三联体），2)同嘌呤单链与同嘌呤-同嘧啶双链之间的三联体DNA （Pry三联体），3)富g和富c单链之间的双联体DNA （Pur双联体），4)富a和富t单链之间的双联体DNA （Pry双联体）。5)富g单链和6)折叠富g单链的四联体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三聚体和Pur双聚体的结合亲和力比与Pur三聚体的结合亲和力小几十倍。STM1不与Pyr双链结合。STM1与富g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 -四聚体表现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三聚体的结合常数小几倍。线性染色体末端端粒区有一部分由富g单链组成，具有形成四联体DNA的潜力。结合目前的结果和端粒区形成四体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端区域可能直接与Less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