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NMR aided structural analysis of Holiday Junction

Holiday Junction 的核磁共振辅助结构分析

基本信息

批准号：
09044098
负责人：
ONO Akira
金额：
$ 5.5万
依托单位：
TOKYO METOROPOLITAN UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044098/
关键词：
Heteronuclear Mutidimensional NMR Stable isotope labeling Chemical Synthesis of DNA Holliday Junction Holliday Junction Holliday junction NMR 安定同位体標識核酸安定同位体利用NMR 安定同位体 DNA 化学合成

项目摘要

The synthetic method for site and stereoselectively ィイD12ィエD1H/ィイD113ィエD1C-labeled glucose had been investigated. After examination of the reaction conditions for introducing deuteron at 5-positions of ィイD113ィエD1C-labeled ribose by the deuteride transfer from deuterated reagent to the 5-oxopentose derivatives synthesized from ィイD113ィエD1C-labeled glucose, the procedure introducing deuteron at the 5-position of ribose with desired R/S ratios. Finally, the synthetic route for site and stereoselectively ィイD12ィエD1H/ィイD113ィエD1C-labeled nucleosides had been established.We examined the reaction conditions for synthesizing labeled dNMP by phosphorylating the 5'OH of labeled nucleosides. The yields for the phosphrylation of 2'-deoxynucleosides are lower than those for the phosphrylation of ribo-nucleosides. However, the yields have been improved by adding protonsponge as a base.The reaction conditions have been investigated for in vitro replication using labeled dNTP and exo-nuclease activity free Klenow polymerase. We found that the commercially available enzyme gave good results.Through NMR studies using ィイD113ィエD1C/ィイD115ィエD1N-fully labeled DNA duplexes, NMR scalar coupling across Watson-Crick base pair hydrogen bonds were observed. This phenomenon can be used for robust determination of base pairs in large complexes such as the Holliday Junction.We tried to express Ruv C, a Holliday Junction specific endonuclease, and its mutants using high expression vectors in order to study Ruv C-Holliday Junction complexes by NMR, however, the purpose has not achieved because of the low solubility of the enzyme.

研究了位点和立体选择性ィイD12ィエD1H/ィイD113ィエD1C标记葡萄糖的合成方法。通过从氘化试剂向由ィイD113ィD1C标记的葡萄糖合成的5-氧代戊糖衍生物进行氘化物转移，对在ィイD113ィD1C标记的核糖的5位导入氘核的反应条件进行了研究，以期望的R/S比在核糖的5位导入氘核。最终建立了位点和立体选择性ィイD12ィエD1H/ィイD113ィエD1C标记核苷的合成路线。我们考察了通过磷酸化标记核苷的5'OH来合成标记dNMP的反应条件。 2'-脱氧核苷磷酸化的产率低于核糖核苷磷酸化的产率。然而，通过添加质子海绵作为碱基，产量得到了提高。使用标记的 dNTP 和无外切核酸酶活性的 Klenow 聚合酶研究了体外复制的反应条件。我们发现市售酶给出了良好的结果。通过使用ィイD113ィエD1C/ィイD115ィエD1N完全标记的DNA双链体进行NMR研究，观察到Watson-Crick碱基对氢键上的NMR标量耦合。这种现象可用于对霍利迪连接点等大型复合物中的碱基对进行稳健测定。我们尝试使用高表达载体表达霍利迪连接点特异性核酸内切酶Ruv C及其突变体，以便通过NMR研究Ruv C-霍利迪连接点复合物，但由于该酶的溶解度较低，未能达到目的。