The Absence of a Pyrimidine Dimer Repair Mechanism in Mammalian Mitochondria

Abstract

We have investigated whether mammalian cells can repair pyrimidine dimers in their mitochondrial DNA which have been induced by ultraviolet light. The assay system is based upon the ability of the phage T4 UV endonuclease to nick covalently closed circular mitochondrial DNA that contain pyrimidine dimers. Our At least three modes of repair of ultraviolet light-damaged DNA exist in prokaryotic cells: photoreactivation, dimer excision repair, and postreplication repair (1-3). In placental mammalian cells, excision repair of nuclear DNA has been demonstrated unequivocally (4) and a recent report suggests the presence of a post-replication repair mechanism (5). Photoreactivation of pyrimidine dimers in nuclear DNA of placental mammals has not been reported (6).Mammalian mitochondria contain their own distinct genetic material in the form of closed circular DNA molecules (7), which are presumably susceptible to the damaging effects of ultraviolet radiation. We were therefore interested in determining whether or not mammalian cells contain a repair mechanism(s) for removal of ultraviolet light-induced pyrimidine dimers in mitochondrial DNA (mtDNA). In addition, recent work has established that these DNA molecules replicate in situ and the mechanism of replication has been described (8-11). If mammalian mitochondria could be shown to carry out repair of damaged DNA, it is possible that some of the previously described presumptive replicating forms of mtDNA might in part be repair intermediates.Abbreviations: EthBr, ethidium bromide; mtDNA, mitochondrial DNA; TK-, thymidine kinase minus; nicked DNA, DNA containing one or more strand breaks; UV, ultraviolet. 2777We have utilized the ability of the pyrimidine dimer-specific phage T4 endonuclease to assay the disappearance of UV light-induced pyrimidine dimers in closed circular mtDNA as a function of time after exposure. This enzyme cleaves a phosphodiester bond near the pyrimidine dimer site; in the case of closed circular DNA, such a cleavage produces a form readily distinguished from the closed circular form by equilibrium centrifugation in EthBr(ethidium bromide)-CsCl density gradients (12). The results of these experiments indicate that mouse L cells, human KB and HeLa cells do not remove pyrimidine dimers from their mtDNA. MATERIALS AND METHODSCells. Mouse L cells, human KB, and human HeLaTKcells were grown on 100-mm Falcon dishes in minimal essential medium plus 10% calf serum as previously described (13). In some experiments we have utilized TK-cells in order to assay specifically for isotopically labeled mtDNA in density gradients (9,10,14). When the cells reached approximately 50% confluence the medium was removed and the cells were washed twice with pre-warmed TD buffer (0.133 M NaCl, 0.005 M KCl, 0.0007 M Na2HPO4, 0.025 M Tris, pH 7.4). Cells were exposed to UV light from a 15-W General Electric low pressure mercury vapor germicidal lamp. UV

Do mitochondrial DNA fragments promote cancer and aging?

“…The steady-state concentration of oxygen radicals may be even higher in tumor mitochondria since they have diminished amounts of superoxide dismutase [53,54] There is a general consensus that mitochondria are less efficient in repairing DNA damage and replication errors than the nucleus [56][57][58][59]. For example, they lack excision repair and recombinational repair [60,61]. Mitochondria do, however, possess three uracil DNA glycosylases [62][63][64], two endonuclease activities specific for apurinic/ apyrimidinic sites [Tomkinson, A.E.…”

A pattern of accumulation of a somatic deletion of mitochondrial DNA in aging human tissues.

“…The accumulation of deleterious mutations in the mitochondrial genome has also been proposed to be important in aging (2)(3)(4)(5). It was known that some types of DNA damage occur at higher levels in mitochondrial DNA (mtDNA) and that mitochondria are deficient for some types of DNA repair; some repeated nuclear sequences also show such a deficiency (11)(12)(13)(14). But assays with the sensitivity to detect the basal level of some types of mitochondrial mutation in normal human tissues have only recently been developed (5,15,16).…”

Mitochondrial DNA repair in aging and disease

“…Such views were based on early experiments showing that UV-induced pyrimidine dimers were not repaired in mtDNA (28). However, subsequent studies have demonstrated that certain types of damage to DNA bases resulting from deamination, simple alkylation and oxidation can be efficiently repaired in mitochondria (reviewed in 29-31).…”