Single-nucleotide patch base excision repair of uracil in DNA by mitochondrial protein extracts.
Mammalian mitochondria contain several 16.5 kb circular DNAs (mtDNA) encoding electron transport chain proteins. Reactive oxygen species formed as byproducts from oxidative phosphorylation in these organelles can cause oxidative deamination of cytosine and lead to uracil in mtDNA. Upon mtDNA replication, these lesions, if unrepaired, can lead to mutations. Until recently, it was thought that there was no DNA repair in mitochondria, but lately there is evidence that some lesions are efficiently repaired in these organelles. In the study of nuclear DNA repair, the in vitro repair measurements in cell extracts have provided major insights into the mechanisms. The use of whole-cell extract based DNA repair methods has revealed that mammalian nuclear base excision repair (BER) diverges into two pathways: the single-nucleotide replacement and long patch repair mechanisms. Similar in vitro methods have not been available for the study of mitochondrial BER. We have established an in vitro DNA repair system supported by rat liver mitochondrial protein extract and DNA substrates containing a single uracil opposite to a guanine. Using this approach, we examined the repair pathways and the identity of the DNA polymerase involved in mitochondrial BER (mtBER). Employing restriction analysis of in vitro repaired DNA to map the repair patch size, we demonstrate that only one nucleotide is incorporated during the repair process. Thus, in contrast to BER in the nucleus, mtBER of uracil in DNA is solely accomplished by single-nucleotide replacement.
Stierum
R H
RH
Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA.
Dianov
G L
GL
Bohr
V A
VA
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D020137:Base Pair Mismatch / Q000235:genetics*; D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism; D000074002:DNA Polymerase gamma; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000235:genetics; D019384:Nucleic Acid Synthesis Inhibitors; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015183:Restriction Mapping; D013696:Temperature; D014500:Uracil Nucleotides / Q000235:genetics* / Q000378:metabolism
D019384:Nucleic Acid Synthesis Inhibitors; D009838:Oligodeoxyribonucleotides; D014500:Uracil Nucleotides; D016590:Aphidicolin; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases; D005033:Ethylmaleimide