Mapping at the nucleotide level of Xenopus laevis mitochondrial D-loop H strand: structural features of the 3' region

The D-loop resulting from limited synthesis of the newly replicated heavy (H) strand of mitochondrial DNA provides a good opportunity to examine both the origin and termination of DNA synthesis. We report here the precise determination of the 3' and 5' termini of nascent Xenopus laevis D-loop H strand. We observe two major classes of newly synthesized D-loop H strands, 1641 and 1675 nucleotides long. A stable putative secondary structure located around its 3' end is described. Analogous secondary structures are also found in the same region of the mammalian D-loop mitochondrial DNAs. Moreover a pentanucleotide (5' TACAT 3'), base-paired in these secondary structures and most often present in two copies, is conserved in all vertebrate species so far studied. The termination associated sequence previously described in mammals is part of the putative stop signal represented by the secondary structure except in man. These results show that the mechanism of arrest of H strand synthesis is common to vertebrates.     

Mechanism of replication of human mitochondrial DNA. Localization of the 5' ends of nascent daughter strands

Human mitochondrial DNA contains two physically separate and distinct origins of DNA replication. The initiation of each strand (heavy and light) occurs at a unique site and elongation proceeds unidirectionally. Animal mitochondrial DNA is novel in that short nascent strands are maintained at one origin (D-loop) in a significant percentage of the molecules. In the case of human mitochondrial DNA, there are three distinct D-loop heavy strands differing in length at the 5' end. We report here the localization of the 5' ends of nascent daughter heavy strands originating from the D-loop region. Analyses of the map positions of 5' ends relative to known restriction endonuclease cleavage sites and 5' end nucleotides indicate that the points of initiation of D-loop synthesis and actual daughter strands are the same. In contrast, the second origin is located two-thirds of the way around the genome where light strand synthesis is presumably initiated on a single-stranded template. Mapping of 5' ends of daughter light strands at this origin relative to known restriction endonuclease cleavage sites reveals two distinct points of initiation separated by 37 nucleotides. This origin is in the same relative genomic position and shows a high degree of DNA sequence homology to that of mouse mitochondrial DNA. In both cases, the DNA region within and immediately flanking the origin of DNA replication contains five tightly clustered tRNA genes. A major portion of the pronounced DNA template secondary structure at this origin includes the known tDNA sequences.     

In vitro replication of heavy strand DNA in permeabilized human mitochondria.

We have characterized some of the experimental conditions that are essential for initiation of human mitochondrial DNA synthesis. Mitochondria were purified from HeLa cells and were permeabilized with Triton X-100. When supplied with rNTPs and dNTPs, the permeabilized mitochondria synthesized nucleic acids that ranged in size from about 600 to 2000 nucleotides. In vitro DNA synthesis occurred on endogenous DNA templates and required a continuous supply of ATP. Analyses of the synthetic products revealed that almost all of them were of heavy-strand sequence and included authentic 7S DNA. Most of the synthetic products had 5' ends that mapped to similar locations as those previously identified for nascent heavy-strand DNA. Identification of these parameters should facilitate our efforts to achieve in vitro replication of heavy-strand mitochondrial DNA.     

Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA.

The sequences of the displacement-loop (D-loop) regions of mitochondrial DNA (mtDNA) from mouse L cells and human KB cells have been determined and provide physical maps to aid in the identification of sequences involved in the regulation of replication and expression of mammalian mtDNA. Both D-loop regions are bounded by the genes for tRNAPhe and tRNAPro. This region contains the most highly divergent sequences in mtDNA with the exceptions of three small conserved sequence blocks near the 5' ends of D-loop strands, a 225 nucleotide conserved sequence block in the center of the D-loop strand template region, and a short sequence associated with the 3' ends of D-loop strands. A sequence similar to that associated with the 3' termini of D-loop strands overlaps one of the conserved sequence blocks near the 5' ends of D-loop strands. The large, central conserved sequence probably does not code for a protein since no open reading frames are discretely conserved. Numerous symmetric sequences and potential secondary structures exist in these sequences, but none appear to be clearly conserved between species.     

Electron microscopic localization of the chloroplast DNA replicative origins in Chlamydomonas reinhardii.

Chloroplast DNA, isolated from a synchronized culture of Chlamydomonas reinhardii, was digested with restriction endonucleases and examined in the electron microscope. Restriction fragments containing displacement loops (D-loop) were photographed and measured to determine the position of replicated sequences in relation to the restriction enzyme sites. D-loops were located at two positions on the physical map of chloroplast DNA. One replication origin was mapped at about 10 kb upstream of the 5' end of a 16s rRNA gene. The second origin was spaced 6. 5kb apart from the first origin and was about 16.5 kb upstream of the same 16s rRNA. Initiations at those two sites were not always synchronized. Replication initiated with the formation of a D-loop resulting from the synthesis of one daughter strand. After a short initial lag phase, corresponding to the synthesis of 350 +/- 130 bp of one daughter strand, DNA synthesis then proceeded in both directions. Both D-loop regions were preferred binding sites of undetermined protein complexes.     

Release of replication termination controls mitochondrial DNA copy number after depletion with 2',3'-dideoxycytidine

Although cellular mitochondrial DNA (mtDNA) copy number varies widely among cell lines and tissues, little is known about the mechanism of mtDNA copy number control. Most nascent replication strands from the leading, heavy-strand origin (O_H) are prematurely terminated, defining the 3′ boundary of the displacement loop (D-loop). We have depleted mouse LA9 cell mtDNA to ~20% of normal levels by treating with 2′,3′-dideoxycytidine (ddC) and subsequently allowed recovery to normal levels of mtDNA. A quantitative ligation-mediated PCR assay was used to determine the levels of both terminated and extended nascent O_H strands during mtDNA depletion and repopulation. Depleting mtDNA leads to a release of replication termination until mtDNA copy number approaches a normal level. Detectable total nascent strands per mtDNA genome remain below normal. Therefore, it is likely that the level of replication termination plays a significant role in copy number regulation in this system. However, termination of D-loop strand synthesis is persistent, indicating formation of the D-loop structure has a purpose that is required under conditions of rapid recovery of depleted mtDNA.     

Isolation and nucleotide sequence of a mouse histidine tRNA gene.

We have sequenced a 1307 base pair mouse genomic DNA fragment which contains a histidine tRNA gene. The sequence of the putative mouse histidine tRNA differs from the published sequence of sheep liver histidine tRNA by a single base change in the D-loop. It does not contain an unpaired 5' terminal G residue, as reported for Drosophila and sheep histidine tRNAs. The gene does not contain introns. The 3' flanking region contains a typical RNA polymerase III termination site of 6 consecutive T residues. 523 residues after the 3' end of the his tRNA coding region, the mouse DNA contains a sequence 72% homologous to part of the consensus sequence of the B1 (alu) family.     

Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region.

A methylation protection assay was used in a novel manner to demonstrate a specific bovine protein-mitochondrial DNA (mtDNA) interaction within the organelle (in organello). The protected domain, located near the D-loop 3' end, encompasses a conserved termination-associated sequence (TAS) element which is thought to be involved in the regulation of mtDNA synthesis. In vitro footprinting studies using a bovine mitochondrial extract and a series of deleted mtDNA templates identified a approximately 48-kDa protein which binds specifically to a single TAS element also protected within the mitochondrion. Because other TAS-like elements located in close proximity to the protected region did not footprint, protein binding appears to be highly sequence specific. The in organello and in vitro data, together, provide evidence that D-loop formation is likely to be mediated, at least in part, through a trans-acting factor binding to a conserved sequence element located 58 bp upstream of the D-loop 3' end.     

Mechanism of mitochondrial DNA replication in mouse L-cells: asynchronous replication of strands, segregation of circular daughter molecules, aspects of topology and turnover of an initiation sequence

Examination of in vivo long-labeled, pulse-labeled and pulse-chase-labeled mitochondrial DNA has corroborated and extended the basic elements of the displacement model of replication. Mitochondrial DNA molecules are shown to replicate an average of once per cell doubling in exponentially growing cultures. Analysis of the separate strands of partially replicated molecules indicates that replication is highly asynchronous with heavy-strand synthesis preceding light-strand synthesis. Native and denatured pulse-labeled replicating molecules exhibit sedimentation properties predicted by the displacement model of replication. Pulse-label incorporated into molecules isolated in the lower band region of ethidium bromide/cesium chloride gradients is found primarily in heavy daughter strands. Pulse-label incorporated into molecules isolated in the upper band region is found primarily in light daughter strands. The results of a series of pulse-chase experiments indicate that the complete process of replication requires approximately 120 minutes. Both daughter molecules are shown to segregate in an open circular form. They are then converted to closed circular molecules having a superhelix density near zero. After closure, the 7 S heavy-strand initation sequence is synthesized, and this process is accompanied by nicking, unwinding and closing of at least one of the parental strands resulting in the formation of the D-loop structure. The 7 S heavy-strand initiation sequence of the D-loop structure is not stable and turns over with a half-life of 7·9 hours. We suggest that all in vivo forms of parental closed circular mitochondrial DNA have superhelix densities of near zero, and that the previously observed superhelix density of closed circular mitochondrial DNA, σ~ ?0·02, results from the loss of the 7 S heavy-strand initiation sequence from D-loop mitochondrial DNA molecules during isolation.