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.     

The excised leader of human cytochrome c oxidase subunit I mRNA which contains the origin of mitochondrial DNA light-strand synthesis accumulates in mitochondria and is polyadenylated.

We identified a polyadenylated RNA species which contains the origin of human mitochondrial DNA light-strand synthesis and the surrounding complementary sequences of the four light-strand-encoded tRNAs. This RNA (RNA 9L) is probably derived from the leader portion of RNA 6 which is excised during the formation of the mature cytochrome c oxidase subunit mRNA (RNA 9). The high degree of secondary structure of this RNA is presumably responsible for its anomalous electrophoretic behavior in denaturing polyacrylamide gels.     

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.     

Precise nucleotide location of the 5' ends of RNA-primed nascent light strands of mouse mitochondrial DNA

The mouse mitochondrial DNA origin of light-strand replication has been defined as a 32-nucleotide region located among five transfer RNA genes in the genomic sequence. A distinctive feature of this origin is its potential to form a perfectly complementary stem and 11-nucleotide loop structure. Previous studies have demonstrated that the 5′ ends of nascent light strands map within this region and a major trinucleotide ribosubstitution site in closed circular mouse mitochondrial DNA has been mapped within the stem sequence.Direct analysis and precise localization of the 5′ ends of nascent light strands indicate that essentially all 5′ ends are ribonucleotides mapping in the originspecific dyadic structure. The major 5′ end identified is the rG at position 5187 in the genomic sequence. Priming of replication most likely occurs within the loop portion of the potential dyad and continues for 2 to 16 nucleotides with a sharply defined switch to deoxyribonucleotide synthesis. This functional transition point is identical in map position to the trinucleotide ribosubstitution site in mature, closed circular mitochondrial DNA.     

Isolation and characterization of a DNA primase from human mitochondria

A family of enzymatic activities isolated from human mitochondria is capable of initiating DNA replication on single-stranded templates. The principal enzymes include at least a primase and DNA polymerase gamma and require that rNTPs as well as dNTPs be present in the reaction mixture. Poly(dC) and poly(dT), as well as M13 phage DNA, are excellent templates for the primase activity. A single-stranded DNA containing the cloned origin of mitochondrial light-strand synthesis can be a more efficient template than M13 phage DNA alone. Primase and DNA polymerase activities were separated from each other by sedimentation in a glycerol density gradient. Using M13 phage DNA as template, these mitochondrial enzymes synthesize RNA primers that are 9 to 12 nucleotides in size and are covalently linked to nascent DNA. The formation of primers appears to be the rate-limiting step in the replication process. Replication of M13 DNA is sensitive to N-ethylmaleimide and dideoxynucleoside triphosphates, but insensitive to rifampicin, alpha-amanitin, and aphidicolin.     

In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.     

Human cytomegalovirus UL84 interacts with an RNA stem-loop sequence found within the RNA/DNA hybrid region of oriLyt

Human cytomegalovirus (HCMV) lytic DNA replication is initiated at the complex cis-acting oriLyt region, which spans nearly 3 kb. DNA synthesis requires six core proteins together with UL84 and IE2. Previously, two essential regions were identified within oriLyt. Essential region I (nucleotides [nt] 92209 to 92573) can be replaced with the constitutively active simian virus 40 promoter, which in turn eliminates the requirement for IE2 in the origin-dependent transient-replication assay. Essential region II (nt 92979 to 93513) contains two elements of interest: an RNA/DNA hybrid domain and an inverted repeat sequence capable of forming a stem-loop structure. Our studies now reveal for the first time that UL84 interacts with a stem-loop RNA oligonucleotide in vitro, and although UL84 interacted with other nucleic acid substrates, a specific interaction occurred only with the RNA stem-loop. Increasing concentrations of purified UL84 produced a remarkable downward-staircase pattern, which is not due to a nuclease activity but is dependent upon the presence of secondary structures, suggesting that UL84 modifies the conformation of the RNA substrate. Cross-linking experiments show that UL84 possibly changes the conformation of the RNA substrate. The addition of purified IE2 to the in vitro binding reaction did not affect binding to the stem-loop structure. Chromatin immunoprecipitation assays performed using infected cells and purified virus show that UL84 is bound to oriLyt in a region adjacent to the RNA/DNA hybrid and the stem-loop structure. These results solidify UL84 as the potential initiator of HCMV DNA replication through a unique interaction with a conserved RNA stem-loop structure within oriLyt.     

Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli.

A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.     

Two novel gene orders and the role of light-strand replication in rearrangement of the vertebrate mitochondrial genome

Two novel mitochondrial gene arrangements are identified in an agamid lizard and a ranid frog. Statistical tests incorporating phylogeny indicate a link between novel vertebrate mitochondrial gene orders and movement of the origin of light-strand replication. A mechanism involving errors in light-strand replication and tandem duplication of genes is proposed for rearrangement of vertebrate mitochondrial genes. A second mechanism involving small direct repeats also is identified. These mechanisms implicate gene order as a reliable phylogenetic character. Shifts in gene order define major lineages without evidence of parallelism or reversal. The loss of the origin of light-strand replication from its typical vertebrate position evolves in parallel and, therefore, is a less reliable phylogenetic character. Gene junctions also evolve in parallel. Sequencing across multigenic regions, in particular transfer RNA genes, should be a major focus of future systematic studies to locate novel gene orders and to provide a better understanding of the evolution of the vertebrate mitochondrial genome.      

Evolutionary Shifts in Three Major Structural Features of the Mitochondrial Genome Among Iguanian Lizards

A phylogenetic tree for major lineages of iguanian lizards is estimated from 1,488 aligned base positions (858 informative) of newly reported mitochondrial DNA sequences representing coding regions for eight tRNAs, ND2, and portions of ND1 and COI. Two well-supported groups are defined, the Acrodonta and the Iguanidae (sensu lato). This phylogenetic hypothesis is used to investigate evolutionary shifts in mitochondrial gene order, origin for light-strand replication, and secondary structure of tRNA^Cys. These three characters shift together on the branch leading to acrodont lizards. Plate tectonics and the fossil record indicate that these characters changed in the Jurassic. We propose that changes to the secondary structure of tRNA^Cys may destroy function of the origin for light-strand replication which, in turn, may facilitate shifts in gene order. Received: 28 May 1996 / Accepted: 27 December 1996