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FUNCTION OF NOVEL MAMMALIAN GLYCOSYLASE FOR OXYDATIVE PYRIMIDINE

新型哺乳动物糖基化酶对氧化嘧啶的作用

基本信息

批准号：
15510059
负责人：
KUBO Kihei
金额：
$ 1.98万
依托单位：
OSAKA PREFECTURE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15510059/
关键词：
DNA REPAIR REACTIVE OXYGEN GLYCOSYLASE BASE DAMAGE AP SITES THYMINE GLYCOL BASE EXCISION REPAIR MAMMALIAN マウスグルコシラーゼ

项目摘要

Reactive oxygen species induced by ionizing radiation and intracellular metabolic process attack DNA bases to create various oxidative damages, such as thymine glycol(TG). These damaged bases are mainly repaired by base excision repair(BER) pathway. The novel monofunctional thymine glycol(TG) activity was extracted from isolated nuclei of mouse stomach and spleen after removing mNTH1 and major AP endonuclease by the high-salt treatment. The western blot analysis revealed that the extract prepared did not include a detectable amount of mNTH1 protein. The nuclear extract was further purified either by the combination of UNO-S1 and UNO-Q1 column or by hydrophobic, hydroxyapatite and UNO-S1 column chromatography. The optimum pH and salt condition of the target activity were 100 mM KCl and pH 8.0, respectively. The activity was resistant to 0.58 mM EDTA. After SDS-PAGE and 2D-electrophesis analyses, we found several candidate protein spots or bands ; 2 major (61,56 kDa) with 6 minor candidates (70,53,37,33,27,25 kDa). We analyzed them with MALDI-FTICR Mass Spectrometry. No known TG-DNA glycosylases (mNth1 and mNei1) were included in the candidate proteins analyzed by Mascot Search. XP_140547, BAB_32018, XP_359164 (NCBI protein data base) were identified in the analysis. These proteins were PCR-cloned and the pET-expression systems were constructed.To analyze the action of the glycosylase, a novel AP site detection method was developed using fluorescein-conjugated ARP (FARP-1). The intracellular AP sites were successfully and quantitatively detected by the method. By the aid of this new method, intranuclear BER reaction was successfully monitored after MMS treatment of HeLa cells.

电离辐射和细胞内代谢过程诱导的活性氧攻击DNA碱基，造成各种氧化损伤，如胸腺嘧啶二醇(Tg)。这些受损的碱基主要通过碱基切除修复(BER)途径修复。用高盐处理去除mNTH1和主要AP内切酶后，从小鼠胃和脾的分离核中提取了新的单功能胸腺嘧啶二醇(TG)活性。蛋白质印迹分析表明，所制备的提取物不包含可检测到的mNTH1蛋白。用UNO-S1和UNO-Q1柱层析或疏水、羟基磷灰石和UNO-S1柱层析进一步纯化核抽提物。其最适pH为100 mM KCl，最适盐条件为pH 8.0。该酶对0.58 mM EDTA具有抗性。经过SDS-PAGE和2D-PAGE分析，我们发现了几个候选蛋白质斑点或条带：2个主要蛋白质(61，56 kDa)和6个次要候选蛋白质(70，53，37，33，27，25 kDa)。我们用MALDI-FTICR质谱仪对它们进行了分析。Mascot Search分析的候选蛋白中没有已知的TG-DNA糖基酶(mNth1和mNei1)。分析中鉴定出XP_140547、BAB_32018、XP_359164(NCBI蛋白质数据库)。为了分析糖基酶的作用，利用荧光素标记的ARP(FARP-1)建立了AP位点的检测方法。该方法成功地对细胞内AP位点进行了定量检测。借助于这种新的方法，成功地监测了MMS处理HeLa细胞后的核内BER反应。