Analysis of oxidative damage of mitochondrial DNA

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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).