Mitochondrial DNA synthesis during the cell cycle of Saccharomyces cerevisiae

Logarithmically growing cultures of Saccharomyces cerevisiae were separated into fractions of increasing average age by zonal centrifugation. Nuclear and mitochondrial DNA labeled for both long and short periods were examined at different times in the cell cycle following isolation of the DNA on CsCl gradients. The data thus obtained were used to determine the relative times of replication. In S. cerevisiae the synthesis of nuclear and mitochondrial DNA was found to occur at the same time in the cell cycle. Some data indicate that mitochondrial DNA synthesis was completed before nuclear DNA synthesis.     

Mitochondrial DNA synthesis in cell cycle mutants of saccharomyces cerevisiae

Mitochondrial DNA replication was examined in mutants for seven different Saccharomyces cerevisiae genes which are essential for nuclear DNA replication. In cdc8 and cdc21, mutants defective in continued replication during the S phase of the cell cycle, mitochondrial DNA replication ceases at the nonpermissive temperature. Replication is temperature sensitive even when these mutants are arrested in the G1 phase of the cell cycle with α factor, a condition where mitochondrial DNA replication continues for the equivalent of several generations at the permissive temperature. Therefore the cessation of replication results from a defect in mitochondrial replication per se, rather than from an indirect consequence of cells being blocked in a phase of the cell cycle where mitochondrial DNA is not normally synthesized. Since the temperature-sensitive mutations are recessive, the products of genes cdc8 and cdc21 must be required for both nuclear and mitochondrial DNA replication. In contrast to cdc8 and cdc21, mitochondrial DNA replication continues for a long time at the nonpermissive temperature in five other cell division cycle mutants in which nuclear DNA synthesis ceases within one cell cycle: cdc4, cdc7, and cdc28, which are defective in the initiation of nuclear DNA synthesis, and cdc14 and cdc23, which are defective in nuclear division. The products of these genes, therefore, are apparently not required for the initiation of mitochondrial DNA replication.     

Probable synchronous replication of mitochondrial DNA in cultures of chick embryo fibroblasts

Cultures of chick embryo fibroblasts were synchronized using a procedure previously described. The profile of incorporation of tritiated thymidine showed a main peak of nuclear DNA replication followed by a small peak between 18 and 24 hr after induction of the cell division, and representing 10 to 25% of the main peak. To identify this small peak, cells were treated with ethidium bromide(EB) chloramphenicol (CAP) or 9-B-D arabinofuranosyl adenine (Ara-A). When EB (1 mug ml-1) and CAP(25mug ml-1) were added at time of induction of mitosis (T0) or 14 hr later (T14) the small peak was suppressed whereas the main peak was not decreased. On the contrary, only the main peak was suppressed when Ara-A was added at T0 or T14. These results suggest that the peak might correspond to the synchronous replication of the mitochondrial DNA during the G2 and M phases of the cell division cycle.     

Effects of Inhibition and Restoration of Protein Synthesis on the Replication of the R Factor Rts1 in Proteus mirabilis

The effect of inhibition of protein synthesis on the replication of the R factor Rts1 in Proteus mirabilis was examined by using the technique of CsCl density gradient centrifugation. Only 12% of the copies of Rts1 were found to replicate during amino acid starvation, whereas there was a 30% increase in the amount of P. mirabilis chromosomal deoxyribonucleic acid (DNA) during the same period. Essentially the same amount of Rts1 and host chromosome replication was observed when chloramphenicol was used to inhibit protein synthesis. The replication of Rts1 DNA was also examined in experiments in which cultures were starved for amino acids in (14)N-labeled medium and then transferred to (15)N-labeled medium containing the required amino acids. These experiments showed that Rts1 replication took place throughout the first generation in (15)N-labeled medium and that each copy of Rts1 was replicated one time during the first generation of chromosomal DNA synthesis in (15)N-medium.     

Replication of nuclear and mitochondrial DNA in X-ray-damaged cells: evidence for a nuclear-specific mechanism that down-regulates replication.

The mechanism by which X rays inhibit DNA replication has been investigated in three distinct populations of DNA molecules in human cells: (a) large chromosomal DNA, (b) a population of 50-100 10.3-kb nuclear episomal plasmids per cell, and (c) a population of about 500 16-kb cytoplasmic mitochondrial DNA molecules per cell. DNA replication was inhibited by X rays in nuclear chromosomal and plasmid DNA, but not in mitochondrial DNA. The mechanism by which ionizing radiation inhibits DNA replication must therefore be nuclear-specific and is unlikely to involve diffusible low-molecular-weight substances. Since mitochondrial DNA exists in the cell as independent 16-kb circular molecules and responds to radiation as would be expected for small targets, the implication for nuclear plasmids is that their replication is regulated by a large target. A current model for DNA replication involves the movement of DNA through replication centers made up of polymerases, helicases, and associated replication enzymes that are attached to a matrix. The difference in the response to X rays between mitochondrial DNA and nuclear plasmid DNA can be explained if nuclear plasmids are tightly associated with chromosomal DNA and attached to the matrix, and are coordinately replicated.     

Mouse L cell mitochondrial DNA molecules are selected randomly for replication throughout the cell cycle

The number of mitochondrial DNA molecules in a cell population doubles at the same rate as the cell generation time. This could occur by a random selection of molecules for replication or by a process that ensures the replication of each individual molecule in the cell. We have investigated the rate at which mouse L cell mitochondrial DNA molecules labeled with 3H-thymidine during one round of replication are reselected for a second round of replication. Mouse L cells were labeled with 3H-thymidine for 2 hr, chased for various periods of time and then labeled with 5-bromodeoxyuridine for 4 hr immediately before mitochondrial DNA isolation. A constant fraction of 3H-thymidine-labeled mitochondrial DNA incorporated 5-bromodeoxyuridine after chase intervals ranging from 1.5-22 hr. This result demonstrates that mitochondrial DNA molecules replicated in a short time interval are randomly selected for later rounds of replication, and that replication of mitochondrial DNA continues throughout the cell cycle in mouse L cells.     

Control of the Cdc6 replication licensing factor in metazoa: The role of nuclear export and the CUL4 ubiquitin ligase

A central requirement to maintain genome stability is that DNA replication must be tightly controlled so that genomic DNA is replicated only once in a single cell cycle. The prevention of DNA re-replication is achieved by restricting the assembly of pre-replicative complexes (pre RCs) to the period prior to S phase, and ensuring that pre-RCs cannot reform during S phase. The regulation of the replication licensing factors Cdt1 and Cdc6 during S phase is critical to prevent the reformation of pre-RCs. In yeast, Cdc6 is degraded during S phase to block DNA re-replication. In mammals, Cdc6 is exported from the nucleus; however, a variable percentage of endogenous Cdc6 remains nuclear throughout S phase. The perdurance of nuclear Cdc6 has led a number of groups to question whether the nuclear export of Cdc6 is relevant in restricting its activity. A recent study in C. elegans shows that the nuclear export of Cdc6 is in fact critical to prevent DNA re-replication. This work also identifies the CUL-4 ubiquitin ligase as a master regulator that controls DNA replication by regulating both Cdt1 and Cdc6 replication licensing factors.     

Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S phase of the cell cycle

Bovine papilloma virus (BPV) replicates as a multicopy nuclear plasmid in mouse fibroblasts. Using fluorescence activated cell sorting and mitotic selection procedures, we show that the replication of BPV occurs throughout S phase of the cell cycle and that replication is confined to S phase. After one round of chromosomal DNA replication, almost one quarter of BPV plasmids have replicated more than once, while a similar number of plasmids have not replicated at all. While multiple forms of BPV exist in the cell, all forms show the same pattern of replication. These results are consistent with a model in which BPV plasmids are chosen at random for replication throughout, and only during, S phase and support the view that the completion of S phase is a specifically activated event in the cell cycle rather than simply the end of one round of chromosomal DNA replication.     

An increase in mitochondrial DNA promotes nuclear DNA replication in yeast

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD⁺-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication.     

Conservative segregation of tetrameric units of H3 and H4 histones during nucleosome replication

We have specifically investigated the behavior of H3 and H4 histones during the replication cycle of MH-134SC cells. Mononucleosomes obtained from cells density-labeled with IdU or dense amino acids in the presence of appropriate radiolabeled precursors were applied to sucrose gradients containing 0.3 M NaCl and 4 M urea for rate zonal centrifugation. This allowed the resolution of dense and normal subnucleosome particles composed of DNA and two molecules each of H3 and H4 without any measurable interparticle histone exchange. On labeling with dense amino acids and radiolabeled lysine, a distinct peak of radiolabeled dense particles was obtained. In contrast, pre-radiolabeled H3 and H4 remained in the normal subnucleosome peak region even after one generation time of culturing with dense amino acids. These data indicate the formation of (H3-H4)2 tetramers composed entirely of new H3 and H4 molecules as well as the conservation of pre-existing tetramers. Density labeling for 1 h with IdU in the presence of radiolabeled lysine yielded a distinct peak of radiolabeled dense particles, indicating the deposition of new tetramers on newly replicated DNA. Similar rate zonal analysis of subnucleosome particles obtained from cells prelabeled for 1 h with radiolabeled lysine followed by various IdU-labeling schedules in nonisotopic media yielded data suggesting that tetramers once deposited do not move about randomly during the replication cycle. A possible mode of nucleosome replication is discussed in the light of the present data.     

Replication of bromodeoxyuridylate-substituted mitochondrial DNA in yeast.

The DNA of several strains of Saccharomyces cerevisiae was labeled by growing the culture in medium supplemented with thymidylate and bromodeoxyuridylate. It was thus possible to follow the course of mitochondrial DNA replication in density shift experiments by determining the buoyant density distribution of unreplicated and replicated DNAs in analytical CsCl gradients. DNA replication was followed for three generations after transfer of cultures from light medium to heavy medium and heavy medium to light medium. Under both conditions, the density shifts observed for mitochondrial DNA were those expected for semiconservative, nondispersive replication. This was further confirmed by analysis of the buoyant density of alkali-denatured hybrid mitochondrial DNA. With this method, no significant recombination between replicated and unreplicated DNA was detected after three generations of growth.     

Nuclear matrix support of DNA replication

In higher eukaryotic cells, DNA is tandemly arranged into 10(4) replicons that are replicated once per cell cycle during the S phase. To achieve this, DNA is organized into loops attached to the nuclear matrix. Each loop represents one individual replicon with the origin of replication localized within the loop and the ends of the replicon attached to the nuclear matrix at the bases of the loop. During late G1 phase, the replication origins are associated with the nuclear matrix and dissociated after initiation of replication in S phase. Clusters of several replicons are operated together by replication factories, assembled at the nuclear matrix. During replication, DNA of each replicon is spooled through these factories, and after completion of DNA synthesis of any cluster of replicons, the respective replication factories are dismantled and assembled at the next cluster to be replicated. Upon completion of replication of any replicon cluster, the resulting entangled loops of the newly synthesized DNA are resolved by topoisomerases present in the nuclear matrix at the sites of attachment of the loops. Thus, the nuclear matrix plays a dual role in the process of DNA replication: on one hand, it represents structural support for the replication machinery and on the other, provides key protein factors for initiation, elongation, and termination of the replication of eukaryotic DNA.     

Synthesis and turnover of Euglena gracilis mitochondrial DNA

Replication of mitochondrial DNA was investigated by a density transfer experiment in a strain of Euglena gracilis lacking chloroplast DNA. DNA was uniformly labeled in a medium containing ³²P-labeled inorganic phosphate and [³H]adenine in the presence of the heavy-density label and transferred to a medium containing ³²P-labeled inorganic phosphate but no [³H]adenine following removal of the heavy-density label. Replication of nuclear DNA within these cells was used as an internal control. The densities and ratios of the peaks of nuclear DNA were those expected for a strict semiconservative replication. In contrast, replication of mitochondrial DNA was dispersive, as illustrated by the following results: (1) both native and denatured mitochondrial DNA exhibited a single density peak at 1.1 and 2.2 cell doublings after the density transfer. (2) The specific activity of ³H-labeled DNA varied across the peak of native or denatured DNA, indicating a heterogeneous population of molecules exhibiting different degrees of density and radioisotope labeling. This dispersive replication could involve either multiple recombination events or extensive turnover of the DNA or a mixture of both. Extensive dispersion of the sample obtained at 1.1 cell doublings after the density transfer is shown by the persistence of the same peak density for duplex DNA reduced to a molecular weight of 6 × 10⁵ by shearing.Two measures of the rate of replication of mitochondrial DNA were obtained from the densities of native duplex DNA and the rate of decrease in ³H-specific activities of duplex DNA during the experiment. The average of these rates indicates that mitochondrial DNA replicates at least 1.5 times as fast as nuclear DNA. Since there is a constant ratio of mitochondrial DNA:nuclear DNA in a logarithmic culture, mitochondrial DNA was calculated to have a half-life of 1.8 cell doublings.     

Mitochondrial DNA synthesis in synchronous cultures of the yeast,Saccharomyces cerevisiae

The synthesis of mitochondrial DNA (mtDNA) has been investigated by three independent methods of analysis during consecutive synchronous cell cycles in the yeast, Saccharomyces cerevisiae. The rates of pulse-label incorporation indicate maximal [³H]adenine uptake into mtDNA at the time of nuclear DNA synthesis. In contrast, the relative concentrations of mtDNA as determined by both the ratio of mtDNA to total cellular DNA and by the kinetics of isotope dilution analysis were found to increase continuously during synchronous growth. We conclude that whereas nuclear DNA replicates discontinuously during the cell cycle, mitochondrial DNA is synthesized continuously during this time. The discontinuous pattern of pulse-label incorporation into mtDNA is not considered to reflect its true mode of replication during the cell cycle.     

Cell-cycle-specific F plasmid replication: regulation by cell size control of initiation.

F plasmid replication during the Escherichia coli division cycle was investigated by using the membrane-elution technique to produce cells labeled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid DNA. The F plasmid replicated, like the minichromosome, during a restricted portion of the bacterial division cycle; i.e., F plasmid replication is cell-cycle specific. The F plasmid replicated at a different time during the division cycle than a minichromosome present in the same cell. F plasmid replication coincided with doubling in the rate of enzyme synthesis from a plasmid-encoded gene. When the cell cycle age of replication of the F plasmid was determined over a range of growth rates, the cell size at which the F plasmid replicated followed the same rules as did replication of the bacterial chromosome--initiation occurred when a constant mass per origin was achieved--except that the initiation mass per origin for the F plasmid was different from that for the chromosome origin. In contrast, the high-copy mini-R6K plasmid replicated throughout the division cycle.     

A moving DNA replication factory in Caulobacter crescentus

The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. Although the newly replicated origin regions of the chromosome are rapidly moved to opposite cell poles by an active process, the replisome appears to be an untethered replication factory that is passively displaced towards the center of the cell by the newly replicated DNA. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation.