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

Animal mitochondrial DNA (mtDNA) maintains a displacement loop (D loop) at the heavy strand origin of replication. These D loops represent sharply limited synthesis ofheavy strands and provide a unique opportunity to examine the termination of DNA synthesis. Direct sizing at the nucleotide level indicates that the 3' ends of D-loop strands of human and mouse mtDNA are discrete and map within three to five nucleotides on the complementary template strand. In the case of human mtDNA, there is a single trinucleotide stop point 51-53 nucleotides downstream from a 15-nucleotide template sequence

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Exhaustive EcoRI digests of circular dimer mitochondrial DNA (mtDNA) from mouse cell lines LD and LDTK- yield two major fragments whose average lengths are slightly smaller than the corresponding fragments of circular monomer mtDNA from mouse LA9 and LMTK- cells. A third fragment approximately 400 nucleotide pairs in length is frequently produced in less than molar yield. Exhaustive EcoRI digests of circular dimer mtDNA from human acute myelogenous leukemic leucocytes yield three major fragments. The presence of mtDNA resistant to cleavage as well as fragments of intermediate sizes indicatesmicroheterogeneity in the genomic positions of EcoRI recognition sequences in both mouse and human circular dimer mtDNA. Analysis of the distribution averages of circular contour lengths indicates microheterogeneity in the sizes of mouse LD and human mtDNAs. The denatured-renatured EcoRI fragments frequently contain a small loop(s) of single-strand DNA as would occur for deletion(s) or addition(s) of single-strand DNA as would occur for deletion(s) or addition(s) of nucleotide sequences in some of the circular dimer molecules.