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Analysis of oxidative damage of mitochondrial DNA

线粒体DNA氧化损伤分析

基本信息

批准号：
09835009
负责人：
KANG Dongchon
金额：
$ 1.92万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09835009/
关键词：
mitochondria aging 8-oxoguanine MutY MutM DNA repair DNA replication LMPCR 活性酸素

项目摘要

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).

为了研究线粒体 DNA 的完整性和氧化损伤，我们开发了一种使用连接介导的聚合酶链反应 (LMPCR) 的新方法。我们可以通过LMPCR特异性扩增具有游离5'端的DNA链。新生链具有游离5'端，这是复制起点。首先，我们通过 LMPCR 以一个碱基分辨率确定了线粒体 DNA 的复制起点 (JBC, 272, 15275)。就线粒体 DNA 而言，起始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，只需确定真正的新生链。我们成功地通过 LMPCR 选择性检测真正的新生链。通过使用这种方法，我们报道了帕金森病诱导剂 I-甲基-4-苯基吡啶鎓离子 (MPP+) 是线粒体 DNA 复制的选择性抑制剂 (JBC, 272, 9605)。接下来，我们开发了用于序列特异性估计的系统利用 LMPCR 和 8-氧代鸟嘌呤识别 DNA 糖基化酶对线粒体 DNA 进行氧化损伤。 MutM和MutY是DNA糖基化酶，它们分别切割含有8-氧代鸟嘌呤：C和8-氧代鸟嘌呤：A的DNA链。用这些酶切割线粒体 DNA 后，切割的链会被扩增并确定切割位点。在这些实验中，我们发现氧化损伤在线粒体 DNA 中以 8-氧鸟嘌呤 : A 形式积累，而不是 8-氧鸟嘌呤 : C 形式（提交中）。