MITOCHONDRIAL DNA DAMAGE AND REPAIR

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Mitochondrial DNA is a frequent target of chemical damage. Base loss, oxidative damage and reaction with carcinogenic compounds such as benzo(a)pyrene and aflatoxin are thought to occur at rates equal to or greater than the corresponding rates of damage to nuclear DNA. Mitochondria are able to repair some types of DNA damage, such as abasic sites and oxidative lesions (e.g., 8-oxo-dG), but probably not others. Very little is known concerning the enzymology of DNA repair in mitochondria. Replication through DNA adducts or attempts at repair may contribute to the incidence of point mutations and deletions observed in a growing list of human diseases. These diseases range from point mutations associated with rare neuromuscular diseases identified with the acronyms MELAS, MERRF, and NARP to large deletions observed in Kearns Sayre Syndrome. Recent studies have found a correlation between mtDNA damage and type 11 diabetes, Parkinson's disease and aging, although more research is required to establish a role for mtDNA damage in the etiology of these conditions. We propose to investigate the effects of specifically localized lesions in DNA templates prepared by our collaborators in this project on DNA replication by the purified mitochondrial DNA polymerase gamma. These studies will employ templates containing abasic sites, 8-oxo-dG, aminofluorene and benzo(a)pyrene adducts. We will also search for accessory factors that may facilitate translesional synthesis by DNA polymerase gamma. We propose experiments to characterize enzymes involved in mtDNA repair and to attempt to reconstitute repair of abasic sites or 8-oxo-dG lesions in mtDNA. Certain lesions, such as cyclobutane pyrimidine dimers in mtDNA are not thought to be repaired efficiently. We will attempt to increase the efficiency of repair by using molecular genetic techniques to direct repair enzymes such as photolyase or T4 endonuclease (denV) to mitochondria.