人线粒体tRNA^(Leu(UUR))基因氧化损伤与修复研究

Oxidative Damage and The Repair Response Under Oxidative Stress in MTTL1 Gene of Human Mitochondrial DNA*

人mtDNA比核DNA更易受到自由基的氧化损伤 ,这些损伤可以被线粒体内的DNA修复机制所修复 ,损伤与修复是决定突变是否产生的两个重要因素 .为了确定氧化损伤与损伤后修复对mtDNA突变的具体影响 ,采用四氧嘧啶处理LO2 细胞 ,这种试剂进入细胞后 ,经氧化还原反应生成的自由基与线粒体自身代谢产生的自由基类似 ,然后观察自由基对细胞mtDNA的氧化损伤与损伤后DNA修复的动力学变化 .由于线粒体的正常功能为修复机制所必需 ,采用MTT细胞活力实验检测不同浓度四氧嘧啶处理下线粒体酶活力 ,发现 9mmol/L四氧嘧啶培养细胞 1h后 ,线粒体琥珀酸脱氢酶功能在撤去药物后 0 ,2 ,8和 2 4h时间点均无明显变化 .提取各组细胞的mtDNA ,用EndoⅢ和Fgp两种酶切除受氧化损伤的核苷酸 ,然后用碱性琼脂糖凝胶电泳分离大小不等的mtDNA ,进行DNA印迹实验 ,地高辛 抗体 碱性磷酸酶系统显色 ,检测完整与断裂的mtDNA量 ,利用Poisson公式 (s=-lnP0 /P ,P0 为未断裂链光密度值 ,P为所有链光密度值总和 )计算一个mtDNA分子的平均损伤频率 ,结果显示 ,9mmol/L四氧嘧啶处理细胞 1h ,链平均损伤频率由对照的 0 11个 /分子增加至 5 6 0个 /分子 ,明显增加了mtDNA上核苷酸的氧化损伤 ,除去药物后 8h ,绝大部分损伤可被修复 ,损伤频率减至

Mitochondrial DNA is more prone to suffering from extensive oxidative damage than nuclear DNA. Base-excision repair which is well established in mitochondria may be involved in the prevention of nucleotides from oxidative damage. It is necessary to consider which severe damage or inadequate repair mainly contributes to the mutations in mtDNA. Human LO2, liver cells were exposed to 9 mmol/L alloxan for 1 h and then incubated in fresh culture media for 0, 2, 8 and 24 h respectively. The frequencies of oxidative base damage in mtDNA were measured by a quantitative Southern blot coupled with digestion by the enzymes endonuclease M and formamidopyrimiding DNA glycosylase. Next, ligation-mediated PCR (LM-PCR) was performed to map damage to specific nucleotides along a similar to 100 bp fragment including MTTL1 gene. The addition of alloxan to cultured human cells increased the rate of oxidative base damage and, by several fold, the lesion frequency in mtDNA. After removal of this DNA damaging agent from culture, the lesion frequency decreased to levels slightly higher than normal at 8 h and returned to normal levels at 24 h. The result of LM-PCR showed 20 hot spots of MTTL1 gene where nucleotides were receiving a majority of damage. The pattern of oxidative damage is like that of point mutation identified in this gene, suggesting the possibility that oxidative damage mainly contributes to the formation of the point mutation in MTTL1 gene.