Analysis of oxidative damage of mitochondrial DNA

为了研究线粒体DNA的完整性和氧化损伤，我们开发了一种新的方法，使用连接介导的聚合酶链反应（LMPCR）。利用LMPCR技术可以特异性地扩增具有游离5'端的DNA链，新生的DNA链具有游离的5'端，这是复制的起点。首先，我们通过LMPCR（JBC，272，15275）以一个碱基分辨率确定线粒体DNA的复制起点。在线粒体DNA的情况下，启动复制的主要部分被过早终止和放弃（复制失败），导致D环链的形成。这种失败的复制使得复制状态的估计变得复杂。为了估计线粒体DNA的复制状态，只有真正的新生链必须确定。利用LMPCR技术成功地检测到了真新生链，并报道了帕金森病诱导剂MPP+对线粒体DNA复制的选择性抑制作用（JBC，272，9605）接下来，我们利用LMPCR和8-oxoguanine-2，建立了线粒体DNA氧化损伤的序列特异性检测系统。识别DNA糖基化酶。MutM和MutY是DNA糖基化酶，其分别切割含有8-氧代鸟嘌呤：C和8 -氧代鸟嘌呤：A的DNA链。在用这些酶切割线粒体DNA后，扩增切割的链并确定切割位点。在这些实验中，我们发现氧化损伤在线粒体DNA中以8-oxoguanine：A形式而不是8-oxoguanine：C形式积累（提交中）。

To investigate the integrity and oxidative damage of mitochondrial DNA, we developed a new method using ligation-mediated polymerase chain reaction (LMPCR). We can specifically amplify DNA strands having free 5' end by LMPCR.The nascent strands have free 5 ends which are the replication origins. First, we determined the replication origins of mitochondrial DNA at one base resolution by LMPCR (JBC, 272, 15275). In the case of mitochondrial DNA, a major part of initiated replication is prematurely terminated and abandoned (abortive replication), resulting in the formation of the D-loop strands. This abortive replication makes the estimation of the replication status complicated. To estimate the replication status of mitochondrial DNA, only the true nascent strands must be determined. We succeeded in the selective detection of true nascent strands by LMPCR.By using this method, we reported that a Parkinson disease-inducing reagent, I-methyl-4-phenylpyridinium ion (MPP+), is a selective inhibitor of the mitochondrial DNA replication (JBC, 272, 9605).Next, we developed the system for sequence-specific estimation of oxidative damage of mitochondrial DNA by taking advantage of LMPCR and 8-oxoguanine-recognizing DNA glycosylases. MutM and MutY are the DNA glycosylases which cleave DNA strands containing 8-oxoguanine : C and 8 -oxoguanine : A, respectively. After the cleavage of mitochondrial DNA with these enzymes, cleaved strands are amplified and the cleavage sites are determined. In these experiments, we found that oxidative lesion accumulates as a 8-oxoguanine : A form but not a 8-oxoguanine : C in mitochondrial DNA (in submission).