| Research Abstract |
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
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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
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