OxyFile #323

Purification of all forms of HeLa cell mitochondrial DNA 
and assessment of damage to it caused by hydrogen peroxide 
treatment of mitochondria or cells.


Author:  Higuchi Y; Linn S

Source:  J Biol Chem 1995 Apr 7; 270(14):7950-6

Abstract:

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.