In vitro replication of human mitochondrial DNA: accurate initiation at the origin of light-strand synthesis

Synthesis of human light-strand mitochondrial DNA was accomplished in vitro using DNA primase, DNA polymerase, and other accessory proteins isolated from human mitochondria. Replication begins with the synthesis of primer RNA on a T-rich sequence in the origin stem-loop structure of the template DNA and absolutely requires ATP. A transition from RNA synthesis to DNA synthesis occurs near the base of the stem-loop structure and a potential recognition site for signaling that transition has been identified. The start sites of the in vitro products were mapped at the nucleotide level and were found to be in excellent agreement with those of in vivo nascent light-strand DNA. Isolated human mitochondrial enzymes recognize and utilize the bovine, but not the mouse, origin of light-strand replication.     

Mechanism of mitochondrial DNA replication in mouse L-cells: localization and sequence of the light-strand origin of replication

In the novel replication mechanism of closed circular mouse L-cell mitochondrial DNA synthesis one strand of the duplex (the heavy-strand) is initiated at a defined origin and proceeds unidirectionally. Synthesis of the complementary light-strand is initiated at a different origin, located approximately two-thirds genome length from the heavy-strand origin, and also proceeds unidirectionally. The initiation of light-strand synthesis does not occur until synthesis of the heavy-strand has extended past the light-strand origin region. One intriguing consequence of this asynchrony is that the heavy-strand origin functions in a DNA duplex, while the light-strand origin functions as a single-stranded template. In order to obtain the precise location of the light-strand origin we have isolated replicative molecules in which light-strand synthesis has begun and subjected them to digestion by a combination of the single-strand specific nuclease S₁ and various restriction cndonucleases. By comparison of the sizes of the duplex fragments thus generated with those produced by cleavage of non-replicating molecules cleaved with the same enzymes we have located the 5′-end of daughter light-strands at a position 55 to 90 nucleotides from a HpaI cleavage site 0.67 genome length from the heavy-strand origin. The nucleotide sequence of a 318-base region surrounding this site, determined by chemical sequencing techniques, possesses the symmetry required for the formation of three hairpin loops. The most striking of these has a stem consisting of 12 consecutive basepairs and a 13-base loop. In the heavy-strand template, this loop contains 11 consecutive thymidine nucleotides. This light-strand origin region has been found to possess a remarkable degree of homology with several other prokaryotic and eukaryotic origin-related sequences, particularly those of the øX174 A region and the simian virus 40 EcoRII G fragment.It has previously been shown that mouse mitochondrial DNA contains alkali-labile sites, which are presumably due to the presence of ribonucleotides incorporated into the DNA. A cluster of sites, representing eight adjacent ribonucleotides, has been located in mature light strands at or near the origin of light-strand synthesis. The retention of ribonucleotides at this specific location may reflect inefficient removal of an RNA primer at the light-strand origin.     

Nucleotide assignment of alkali-sensitive sites in mouse mitochondrial DNA

Mature, closed circular mouse mitochondrial DNA contains a significant number of ribonucleotides throughout the genome. Previous studies have implicated the two origins of DNA replication as preferred sites of ribonucleotide retention. We have analyzed the site specificity of ribosubstitution by direct sizing of alkali-treated restriction fragments in comparison with the DNA sequence of untreated restriction fragments of cloned mouse mitochondrial DNA. These results have confirmed the observations that ribonucleotides are retained at the two origins of replication and are most likely remnants of RNA priming events associated with DNA replication. The map location of ribonucleotides at the light strand origin of replication has been refined to a triplet nucleotide (5'-CGG-3') in the light strand initiation region. This approach has demonstrated that all four deoxyribonucleotides are subject to ribosubstitution and no single base (or subset of the four bases) predominates. An examination of selected regions of the mitochondrial DNA genome including the putative coding region for cytochrome oxidase subunit III and regions containing the genes for tRNAPhe, tRNAVal, 12 S rRNA, and 16 S rRNA reveals preferred sites for ribosubstitution. These preferred sites do not relate in any obvious way to the functional aspects of these domains. In addition, the data indicate that every position in the DNA sequences examined can be ribosubstituted at a very low frequency.     

Mapping initiation sites for simian virus 40 DNA synthesis events in vitro.

Primer RNA-DNA, a small (approximately 30-nucleotide) RNA-DNA hybrid molecule, was identified in recent studies of simian virus 40 DNA synthesis in vitro. The available evidence indicates that primer RNA-DNA is the product of the polymerase alpha-primase complex. Primer RNA-DNA is formed exclusively on lagging-strand DNA templates; it is synthesized initially in the vicinity of the simian virus 40 origin and at later times at sites progressively distal to the origin. To further characterize initiation events, template sequences encoding the 5' ends of both primer RNA and primer DNA, formed during a 5-s pulse, have been determined. Analyses of these sequences demonstrate the existence of an initiation signal for lagging-strand synthesis. At any given position, the initiation signal is located within those template sequences encoding primer RNA, situated proximal to the nucleotide encoding the 5' end of the RNA primer. In most instances, the sequence 5'-TTN-3' (where N encodes the nucleotide at the 5' end of the primer) is a feature of the initiation signal. Initiation signals are present, on average, once every 19 nucleotides. These results are discussed in terms of the mechanism of Okazaki fragment formation and possible links between prokaryotic and eukaryotic initiation events.