Single-nucleotide patch base excision repair of uracil in DNA by mitochondrial protein extracts

Mammalian mitochondria contain several 16.5 kb circular DNAs (mtDNA) encodi ng electron transport chain proteins. Reactive oxygen species formed as byp roducts from oxidative phosphorylation in these organelles can cause oxidat ive deamination of cytosine and lead to uracil in mtDNA, Upon mtDNA replica tion, these lesions, if unrepaired, can lead to mutations, Until recently, it was thought that there was no DNA repair in mitochondria, but lately the re is evidence that some lesions are efficiently repaired in these organell es, In the study of nuclear DMA 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 nu clear base excision repair (BER) diverges into two pathways: the single-nuc leotide replacement and long patch repair mechanisms. Similar in vitro meth ods have not been available for the study of mitochondrial BER. We have est ablished 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 ide ntity of the DNA polymerase involved in mitochondrial BER (mtBER), Employin g restriction analysis of in vitro repaired DNA to map the repair patch siz e, we demonstrate that only one nucleotide is incorporated during the repai r process, Thus, in contrast to BER in the nucleus, mtBER of uracil in DNA is solely accomplished by single-nucleotide replacement.