Structural morphorism of DNA triplexes and triplet repeat sequences

分子间DNA三链体的形成及化学修饰的影响：影响DNA三链体形成的因素很多，如pH、温度、序列错配、化学修饰等，我们通过等温量热仪和表面等离子体共振方法获得了热力学和动力学参数，并对获得的参数进行了分析，以评价嘧啶第三链的化学修饰的影响。具有dU、RNA和2'-O-甲基的核苷酸之间的熵变化相似。相比之下，与具有化学修饰的碱基或糖部分的其他核苷酸相比，被硫代磷酸酯取代的磷酸骨架在缔合常数方面显著地不稳定，其数量级为一个数量级。这一结果表明，骨架磷酸盐是控制DNA三链体稳定性的最敏感基团。特定DNA序列对染色质结构的影响：许多特定的DNA序列往往导致DNA的高级结构，即所谓的非B DNA的形成。这种特定的序列经常在真核细胞中发现，但它们的生物学作用尚不清楚。我们已经构建了一个测定系统，以检查DNA序列对酵母细胞中的核小体组织的影响，并证明了长道的多聚dA和Z-DNA形成序列被完全排除在一个定位的核小体之外，而三链体形成序列不会破坏，但会使染色质组织不稳定。使用这种微型染色体分析系统，我们研究了三重重复序列如（CTG）n和（CGC）n的影响，表明前者不影响核小体结构，但后者破坏核小体定位。

Formation of intermolecular DNA triplexes and the effects of chemical modifications : There are many causes such as pH, temperature, mismatched sequences, chemical modifications and so on. We obtained thermodynamic and kinetic parameters by an isothermal calorimeter and surface plasmon resonance method, and analyzed obtained parameters to evaluate the effects of chemical modifications of the third pyrimidine strands. Entalpy changes are similar among nucleotides with dU, RNA and 2'-O-methyl. By contrast, phosphate backbone substituted by phosphorothioate was dramatically unstable by an order of the magnitude in terms of association constant, compared with other nucleotides with chemically modified bases or sugar moiety. This result shows that backbone phosphate is most sensitive moiety to control the stability of DNA triplexes.Effects of specific DNA sequences on chromatin structures : Many specific sequences often result in the formation of higher order structures of DNA, so-called non-B DNA. Such specific sequences are frequently found in eucaryotic cells, but their biological roles are not known. We have constructed an assay system to examine effects of DNA sequences on nucleosome organization in yeast cells and demonstrated that long tract of poly dA and Z-DNA forming sequence are completely excluded from a positioned nucleosome, whereas triplex forming sequences do not disrupt but destabilizes chromatin organization. Using this mini chromosome assay system we investigated effects of triplet repeat sequences such as (CTG)n and (CGC)n, showing that the former does not affect the nucleosome structure but the latter disrupts nucleosome positioning.