Purification of all forms of HeLa cell mitochondrial DNA and assessment of damage to it caused by hydrogen peroxide treatment of mitochondria or cells.
A purification scheme for mitochondrial DNA (mtDNA) was designed which maximized the yield of all forms of the DNA while minimizing damage to the DNA during its isolation. Treatment of intact mitochondria with DNase I removed nuclear DNA and the avoidance of phenol and the isolation by CsCl density gradients in the absence of ethidium bromide and subsequent detection by Southern Hydridization dot-blots minimized DNA damage. Four different mtDNA forms free of apparent nuclear DNA were obtained: closed circular (I), open circular (II), linear (III), and a large multimer complex (C) which were characterized by agarose gel electrophoresis and electron microscopy. Using this procedure, mtDNA was obtained from both whole cells or intact mitochondria treated with H2O2. Significant fragmentation was observed after treatment at 37 degrees C, but not at 0 degrees C, and more damage was observed when treating whole cells than isolated mitochondria. Very low levels of 8-hydroxydeoxyguanosine were observed in all cases. However, at doses of H2O2 which were just lethal, neither increased DNA damage nor inactivation of cytochrome c oxidase was observed.
Higuchi
Y
Y
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720-3202, USA.
Linn
S
S
D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000187:drug effects / Q000302:isolation & purification*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects; D008969:Molecular Sequence Data; D018384:Oxidative Stress
D017931:DNA Primers; D004272:DNA, Mitochondrial; D006861:Hydrogen Peroxide