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.     

Initiation of RNA-primed DNA synthesis in vitro by DNA polymerase alpha-primase.

The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-primase complex (pol alpha-primase), a natural DNA template containing a site for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex DNA molecule with an open helix replication 'bubble' to serve as an initiation zone. Pol alpha-primase recognizes the open helix region and initiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ssDNA-dsDNA junction in the replication 'bubble' template is the preferred site for initiation. Using a 40 base oligonucleotide template containing the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC-3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the open helix structure of the replication 'bubble' directs pol alpha-primase to initiate RNA primer synthesis near the ssDNA-dsDNA junction.     

Both the conserved stem-loop and divergent 5'-flanking sequences are required for initiation at the human mitochondrial origin of light-strand DNA replication

Mammalian mitochondrial DNAs contain a conserved origin of light-strand replication that supports accurate initiation of DNA synthesis in vitro. This provides an opportunity to examine the sequence requirements for initiation through in vitro analysis of a series of deleted and mutagenized DNA templates. These assays use enzymes isolated from human mitochondria and single-stranded DNA templates containing deletions or substitutions in the known origin region. The data indicate that accurate and efficient light-strand replication in vitro requires the previously identified stem-loop structure located within a tRNA cluster. In addition, the template sequence 3'-GGCCG-5', located immediately adjacent to the stem, is necessary for efficient replication. This sequence, the complement of which encodes the 3' end of tRNACys, may be the site of transition from RNA primer synthesis to DNA synthesis. Surprisingly, substitutions within a region located in the loop of this origin do not reduce levels of replication.