D'Ham C, Romieu A, Jaquinod M, Gasparutto D, Cadet J
Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, Département de Recherche Fondamentale sur la Matière Condensée, CEA Grenoble, France.
Biochemistry. 1999 Mar 16;38(11):3335-44. doi: 10.1021/bi981982b.
Oligonucleotides that contain a single modified pyrimidine, i.e., thymine glycol (Tg), 5,6-dihydrothymine (DHT), and 5-hydroxycytosine (5-OHC) were synthesized in order to investigate the substrate specificity and the excision mechanism of two Escherichia coli repair enzymes: endonuclease III and formamidopyrimidine DNA glycosylase (Fpg). Three techniques of analysis were employed. A gas chromatography-mass spectrometry (GC-MS) assay with HPLC prepurification was used to quantify the release of the modified bases, while polyacrylamide gel electrophoresis and matrix-assisted laser-desorption ionization-mass spectrometry (MALDI-MS) provided insights into the mechanism of oligonucleotide cleavage. Values of Vm/Km constants lead to the conclusion that the substrates are processed by endonuclease III with the following preference: Tg >> 5-OHC > DHT. This confirms that Tg is an excellent substrate for endonuclease III. Fpg-mediated cleavage of the 5-OHC-containing oligonucleotide is processed at the same rate than endonuclease III. Furthermore, Fpg was found to have a little but relevant activity on DHT-containing oligonucleotide, thus broadening the substrate specificity of this enzyme to a new modified pyrimidine. While 5-OHC-containing oligonucleotides are cleaved by the two enzymes, no or a small amount of the modified base was found to be released, as determined by GC-MS. From these data it may be suggested that 5-OHC could be modified during its enzymatic excision. Finally, MALDI-MS analyses shed new light on the mechanism of action of endonuclease III: the molecular masses of the repaired fragments of 5-OHC- and DHT-containing oligonucleotides showed that endonuclease III cleaves the DNA backbone mainly through a hydrolytic process and that no beta-elimination product was detected.
为了研究两种大肠杆菌修复酶——内切核酸酶III和甲酰胺嘧啶DNA糖基化酶（Fpg）的底物特异性和切除机制，合成了含有单个修饰嘧啶的寡核苷酸，即胸腺嘧啶二醇（Tg）、5,6-二氢胸腺嘧啶（DHT）和5-羟基胞嘧啶（5-OHC）。采用了三种分析技术。使用经高效液相色谱预纯化的气相色谱-质谱（GC-MS）分析法对修饰碱基的释放进行定量，而聚丙烯酰胺凝胶电泳和基质辅助激光解吸电离质谱（MALDI-MS）则有助于深入了解寡核苷酸切割机制。Vm/Km常数的值得出结论，内切核酸酶III处理底物的偏好顺序如下：Tg >> 5-OHC > DHT。这证实了Tg是内切核酸酶III的优良底物。Fpg介导的含5-OHC寡核苷酸的切割与内切核酸酶III的切割速率相同。此外，发现Fpg对含DHT的寡核苷酸有少量但相关的活性，从而将该酶的底物特异性扩展到一种新的修饰嘧啶。虽然含5-OHC的寡核苷酸被这两种酶切割，但通过GC-MS测定发现没有或仅有少量修饰碱基被释放。从这些数据可以推测，5-OHC在其酶促切除过程中可能被修饰。最后，MALDI-MS分析为内切核酸酶III的作用机制提供了新的线索：含5-OHC和DHT的寡核苷酸修复片段的分子量表明，内切核酸酶III主要通过水解过程切割DNA主链，未检测到β-消除产物。
