Elongation of displacement-loop strands in human and mouse mitochondrial DNA is arrested near specific template sequences.

Animal mitochondrial DNA (mtDNA) maintains a displacement loop (D loop) at the heavy strand origin of replication. These D loops represent sharply limited synthesis of heavy 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 (3'T-A-A-C-C-C-A-A-A-A-A-T-A-C-A 5') which is repeated four times in the mouse mtDNA D-loop region 3'(T-A-A-Py-Py-A-A-A-T-T-A-C-A 5'). The stop points of the five major mouse D-loop strands are 24-63 nucleotides downstream from the four repeated template sequences. These results suggest that the arrest of D-loop strand elongation is an event determined by template sequence.

Microheterogeneity detected in circular dimer mitochondrial DNA.

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.

Absence of a pyrimidine dimer repair mechanism for mitochondrial DNA in mouse and human cells.

We have assayed the ability of mammalian cells to remove pyrimidine dimers from their mitochondrial DNA. The results indicate that pyrimidine dimers are not repaired for as long as 48 hr after UV irradiation. Furthermore, molecules containing pyrimidine dimers are unable to replicate and are simply diluted out in subsequent cell divisions.

The absence of a pyrimidine dimer repair mechanism in mammalian mitochondria.

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 results show that dimers are not removed from the mitochondrial DNA of mouse L cells or human KB and HeLa cells. There is also no evidence for photoreactivation of mitochondrial DNA. Analyses of ethidium bromide-cesium chloride equilibrium density gradients of mitochondrial DNA isotopically labeled before and after exposure to ultraviolet light show that the total amount of DNA replication is depressed after exposure. In addition, an increase in the frequency of molecules banding at a position expected for intermediate replicating forms and open circular daughter molecules suggests that the rate of replication is slower (or arrested) in molecules with pyrimidine dimers. The absence of a significant amount of mixing of label incorporated before and after ultraviolet-irradiation is evidence against the occurrence of a large amount of genetic exchange between mitochondrial DNA molecules under these conditions.