Doi Yusuke, Katafuchi Atsushi, Fujiwara Yoshie, Hitomi Kenichi, Tainer John A, Ide Hiroshi, Iwai Shigenori
Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan.
Nucleic Acids Res. 2006 Mar 17;34(5):1540-51. doi: 10.1093/nar/gkl061. Print 2006.
Endonuclease III (Endo III) is a base excision repair enzyme that recognizes oxidized pyrimidine bases including thymine glycol. This enzyme is a glycosylase/lyase and forms a Schiff base-type intermediate with the substrate after the damaged base is removed. To investigate the mechanism of its substrate recognition by X-ray crystallography, we have synthesized oligonucleotides containing 2'-fluorothymidine glycol, expecting that the electron-withdrawing fluorine atom at the 2' position would stabilize the covalent intermediate, as observed for T4 endonuclease V (Endo V) in our previous study. Oxidation of 5'- and 3'-protected 2'-fluorothymidine with OsO4 produced two isomers of thymine glycol. Their configurations were determined by NMR spectroscopy after protection of the hydroxyl functions. The ratio of (5R,6S) and (5S,6R) isomers was 3:1, whereas this ratio was 6:1 in the case of the unmodified sugar. Both of the thymidine glycol isomers were converted to the corresponding phosphoramidite building blocks and were incorporated into oligonucleotides. When the duplexes containing 2'-fluorinated 5R- or 5S-thymidine glycol were treated with Escherichia coli endo III, no stabilized covalent intermediate was observed regardless of the stereochemistry at C5. The 5S isomer was found to form an enzyme-DNA complex, but the incision was inhibited probably by the fluorine-induced stabilization of the glycosidic bond.
核酸内切酶III（Endo III）是一种碱基切除修复酶，可识别包括胸腺嘧啶乙二醇在内的氧化嘧啶碱基。这种酶是一种糖基化酶/裂合酶，在去除受损碱基后与底物形成席夫碱型中间体。为了通过X射线晶体学研究其底物识别机制，我们合成了含有2'-氟胸腺嘧啶乙二醇的寡核苷酸，期望2'位的吸电子氟原子能够稳定共价中间体，正如我们之前研究中观察到的T4核酸内切酶V（Endo V）那样。用OsO4氧化5'-和3'-保护的2'-氟胸腺嘧啶产生了胸腺嘧啶乙二醇的两种异构体。在保护羟基官能团后，通过核磁共振光谱确定了它们的构型。（5R,6S）和（5S,6R）异构体的比例为3:1，而在未修饰糖的情况下，该比例为6:1。两种胸腺嘧啶乙二醇异构体均转化为相应的亚磷酰胺砌块，并被掺入寡核苷酸中。当用大肠杆菌内切酶III处理含有2'-氟化5R-或5S-胸腺嘧啶乙二醇的双链体时，无论C5处的立体化学如何，均未观察到稳定的共价中间体。发现5S异构体形成酶-DNA复合物，但切口可能因氟诱导的糖苷键稳定而受到抑制。
