FUNCTION OF NOVEL MAMMALIAN GLYCOSYLASE FOR OXYDATIVE PYRIMIDINE

电离辐射和细胞内代谢过程诱导的活性氧攻击DNA碱基，产生各种氧化损伤，如胸腺嘧啶乙二醇（TG）。这些受损的碱基主要通过碱基切除修复（BER）途径修复。通过高盐处理去除mNTH1和主要AP内切酶后，从小鼠离体胃和脾核中提取新的单功能胸腺嘧啶乙二醇（TG）活性。western blot分析表明，制备的提取物不含可检测量的mNTH1蛋白。采用UNO-S1柱和UNO-Q1柱联合或疏水、羟基磷灰石柱和UNO-S1柱层析进一步纯化核提取物。目标活性的最佳pH和盐条件分别为100 mM KCl和pH 8.0。其抗EDTA活性为0.58 mM。经过SDS-PAGE和2d - electroesis分析，我们发现了几个候选的蛋白点或带；2个主要候选人（61,56 kDa）， 6个次要候选人（70,53,37,33,27,25 kDa）。我们用MALDI-FTICR质谱分析它们。Mascot Search分析的候选蛋白中没有已知的TG-DNA糖基酶（mNth1和mNei1）。在分析中鉴定出XP_140547、BAB_32018、XP_359164 （NCBI蛋白数据库）。对这些蛋白进行pcr克隆并构建pet表达体系。为了分析糖基化酶的作用，建立了荧光素共轭ARP （FARP-1）检测AP位点的新方法。该方法成功地定量检测了细胞内AP位点。利用这种新方法，成功地监测了MMS处理HeLa细胞后的核内BER反应。

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.