NMR aided structural analysis of Holiday Junction

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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.