Analysis of oxidative damage of mitochondrial DNA

为了研究线粒体DNA的完整性和氧化损伤，我们建立了一种新的连接介导的聚合酶链式反应(LMPCR)方法。我们可以通过LMPCR特异性地扩增具有5‘端的DNA链，新生的链具有5端的自由端，是复制的起点。首先，我们用LMPCR(JBC，272,15275)在一个碱基分辨率下确定了线粒体DNA的复制起点。在线粒体DNA的情况下，启动的复制的主要部分被过早终止和放弃(中止复制)，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。要估计线粒体DNA的复制状态，必须只确定真正的新生链。我们成功地用LMPCR选择性地检测了真正的新生DNA链。通过使用这种方法，我们报道了一种帕金森病诱发试剂I-甲基-4-苯基吡啶离子(MPP+)是线粒体DNA复制的选择性抑制物(JBC，272,9605)。接下来，我们利用LMPCR和8-氧鸟嘌呤识别DNA糖基酶的优势，建立了线粒体DNA氧化损伤的序列特异性评估系统。MutM和MutY是分别切割含有8-氧鸟嘌呤：C和8-氧鸟嘌呤：A的DNA糖基酶。线粒体DNA被这些酶切割后，被切割的链被扩增并确定切割位置。在这些实验中，我们发现氧化损伤以8-氧鸟嘌呤的形式积累：在线粒体DNA中是一种形式，但不是8-氧鸟嘌呤：C(提交)。

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).