A question of time: replication origins of eukaryotic chromosomes.
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Animals↗
Biomedical subjects
W L Fangman
Publications and source records attributed to W L Fangman.
The arrest of replication forks in the rDNA of yeast occurs independently of transcription.
Replication forks, moving opposite to the direction of transcription, are arrested at the 3' ends of the 35S transcription units in the rDNA locus of S. cerevisiae. Because of its position and polarity, we tested the hypothesis that this replication fork barrier (RFB) results from the act of transcription. Three results contradict this hypothesis. First, the RFB persists in a strain containing a disruption of the gene for the 135 kd subunit of RNA polymerase I. Second, the RFB causes a polar arrest of replication forks when transplanted to a plasmid. Third, transcription by RNA polymerase II of a plasmid copy of the 35S transcription unit lacking the RFB does not generate a barrier. We propose that replication forks are arrested in a directional manner through the binding of one or more proteins to two closely spaced sites in the RFB.
DNA Replication↗
A position effect on the time of replication origin activation in yeast.
The chromosomes of eukaryotes are characterized by the mosaic nature of their replication--large regions of DNA that replicate early in S phase are interspersed with regions that replicate late. This pattern of early and late synthesis appears to be the consequence of a temporal program that activates replication origins at different times. The basis of this temporal regulation in the yeast S. cerevisiae has been investigated by changing the chromosomal locations of two origins, one activated early in the S phase (ARS1) and one activated late (ARS501). We show that the cis-acting information controlling time of activation can be separated from the element that determines origin function. For the ARS501 origin, late activation appears to be a consequence of its proximity to the telomere.
Chromosomes, Fungal↗
Mitochondrial DNA maintenance in yeast requires a protein containing a region related to the GTP-binding domain of dynamin.
Nuclear gene products replicate and partition mitochondrial DNA (mtDNA) molecules in the yeast Saccharomyces cerevisiae. However, few of the relevant genes have been identified. A mutation that results in temperature-sensitive loss of mtDNA identifies one of these genes, MGM1. Deletion of MGM1 shows that aside from its role in the mitochondrion, the gene has no essential cellular function. The MGM1 protein has a 200-amino-acid region that is highly related to a family of GTP-binding proteins of apparently diverse function that includes the microtubule-binding protein, dynamin D100. The temperature-sensitive strain partitions mtDNA molecules at the restrictive temperature, but a defect in mtDNA synthesis results in a reduction in the number of molecules per cell at each cell division. On the basis of the results of this study, we conclude that cells can partition single mitochondrial genomes, and that when a cell receives a single molecule at division it is able to restore the normal complement of multiple copies.
Amino Acid Sequence↗
A yeast origin of replication is activated late in S phase.
The mechanism that causes large regions of eukaryotic chromosomes to remain unreplicated until late in S phase is not understood. We have found that 67 kb of telomere-adjacent DNA at the right end of chromosome V in S. cerevisiae is replicated late in S phase. An ARS element in this region, ARS501, was shown by two-dimensional gel analysis to be an active origin of replication. Kinetic analyses indicate that the rate of replication fork movement within this late region is similar to that in early replicating regions. Therefore, the delayed replication of the region is a consequence of late origin activation. The results also support the idea that the pattern of interspersed early and late replication along the chromosomes of higher eukaryotes is a consequence of the temporal regulation of origin activation.
Base Sequence↗
Mapping replication origins in yeast chromosomes.
The replicon hypothesis, first proposed in 1963 by Jacob and Brenner, states that DNA replication is controlled at sites called origins. Replication origins have been well studied in prokaryotes. However, the study of eukaryotic chromosomal origins has lagged behind, because until recently there has been no method for reliably determining the identity and location of origins from eukaryotic chromosomes. Here, we review a technique we developed with the yeast Saccharomyces cerevisiae that allows both the mapping of replication origins and an assessment of their activity. Two-dimensional agarose gel electrophoresis and Southern hybridization with total genomic DNA are used to determine whether a particular restriction fragment acquires the branched structure diagnostic of replication initiation. The technique has been used to localize origins in yeast chromosomes and assess their initiation efficiency. In some cases, origin activation is dependent upon the surrounding context. The technique is also being applied to a variety of eukaryotic organisms.
Blotting, Southern↗
A nuclear mutation reversing a biased transmission of yeast mitochondrial DNA.
The highly biased transmission of p- mitochondrial DNA that occurs in hypersuppressive matings between p- and p+ cells of the yeast Saccharomyces cerevisiae is thought to be a consequence of the replication advantage of the p- mtDNA. A nuclear gene, MGT1, that is required for this displacement of p+ mtDNA from zygotic clones has been identified through mutation. When one haploid parent carries the mgt1 allele, transmission of p- mtDNA is substantially reduced. When both haploid parents carry the mgt1 allele, p- mtDNA is essentially eliminated from the zygotic progeny. Thus in the absence of the MGT1 gene there is a switch in the transmission bias; p+ mtDNA rather than the hypersuppressive p- mtDNA is inherited by most zygotic clones. In contrast to its semi-dominant behavior in haploid matings, mgt1 behaves as a recessive allele in diploid matings since the p+ genome in MGT1/mgt1 diploids is efficiently displaced when mated with a MGT1/mgt1 hypersuppressive p- diploid strain. We find that p+ genomes can be comaintained along with hypersuppressive p- mtDNA for extended periods in clonal lines derived from MGT1 x mgt1 matings. However, as expected from the recessive nature of the mgt1 mutation, these p+ genomes are eventually eliminated. Our work indicates that MGT1 plays a crucial role in the competition for inheritance between hypersuppressive p- mtDNAs and the p+ mitochondrial genome. The MGT1 gene product may be a component of a mtDNA replication system that acts preferentially at the rep sequences found in hypersuppressive mtDNAs.
Cell Nucleus↗
Temporal control of DNA replication in yeast.
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Chromosome Mapping↗
Activation of replication origins within yeast chromosomes.
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Base Sequence↗
RPO41-independent maintenance of [rho-] mitochondrial DNA in Saccharomyces cerevisiae.
A subset of promoters in the mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae has been proposed to participate in replication initiation, giving rise to a primer through site-specific cleavage of an RNA transcript. To test whether transcription is essential for mtDNA maintenance, we examined two simple mtDNA deletion ([rho-]) genomes in yeast cells. One genome (HS3324) contains a consensus promoter (ATATAAGTA) for the mitochondrial RNA polymerase encoded by the nuclear gene RPO41, and the other genome (4a) does not. As anticipated, in RPO41 cells transcripts from the HS3324 genome were more abundant than were transcripts from the 4a genome. When the RPO41 gene was disrupted, both [rho-] genomes were efficiently maintained. The level of transcripts from HS3324 mtDNA was decreased greater than 400-fold in cells carrying the RPO41 disrupted gene; however, the low-level transcripts from 4a mtDNA were undiminished. These results indicate that replication of [rho-] genomes can be initiated in the absence of wild-type levels of the RPO41-encoded RNA polymerase.
Base Sequence↗
Time of replication of ARS elements along yeast chromosome III.
The replication of putative replication origins (ARS elements) was examined for 200 kilobases of chromosome III of Saccharomyces cerevisiae. By using synchronous cultures and transfers from dense to light isotope medium, the temporal pattern of mitotic DNA replication of eight fragments that contain ARSs was determined. ARS elements near the telomeres replicated late in S phase, while internal ARS elements replicated in the first half of S phase. The results suggest that some ARS elements in the chromosome may be inactive as replication origins. The actively expressed mating type locus, MAT, replicated early in S phase, while the silent cassettes, HML and HMR, replicated late. Unexpectedly, chromosome III sequences were found to replicate late in G1 at the arrest induced by the temperature-sensitive cdc7 allele.
Chromosome Mapping↗
A yeast mutation that stabilizes a plasmid bearing a mutated ARS1 element.
To identify the trans-acting factors involved in autonomously replicating sequence (ARS) function, we initiated a screen for Saccharomyces cerevisiae mutants capable of stabilizing a plasmid that contains a defective ARS element. The amm (altered minichromosome maintenance) mutations recovered in this screen defined at least four complementation groups. amm1, a mutation that has been studied in detail, gave rise to a 17-fold stabilization of one defective ARS1 plasmid over the level seen in wild-type cells. The mutation also affected the stability of at least one plasmid bearing a wild-type ARS element. amm1 is an allele of the previously identified TUP1 gene and exhibited the same pleiotropic phenotypes as other tup1 mutants. Plasmid maintenance was also affected in strains bearing a TUP1 gene disruption. Like the amm1 mutant, the tup1 disruption mutant exhibited ARS-specific plasmid stabilization; however, the ARS specificities of these two mutants differed. The recovery of second-site mutations that suppressed many of the tup1 phenotypes but not the increased plasmid maintenance demonstrates that the plasmid stability phenotype of tup1 mutants is not a consequence of the other defects caused by tup1.
Chromosome Mapping↗
Stable maintenance of a 35-base-pair yeast mitochondrial genome.
Small deletion variants ([rho-] mutants) derived from the wild-type ([ rho+]) Saccharomyces cerevisiae mitochondrial genome were isolated and characterized. The mutant mitochondrial DNAs (mtDNAs) examined retained as little as 35 base pairs of one section of intergenic DNA, were composed entirely of A.T base pairs, and were stably maintained. These simple mtDNAs existed in tandemly repeated arrays at an amplified level that made up approximately 15% of the total cellular DNA and, as judged by fluorescence microscopy, had a nearly normal mitochondrial arrangement throughout the cell cytoplasm. The simple nature of these [rho-] genomes indicates that the sequences required to maintain mtDNA must be extremely simple.
Base Sequence↗
A replication fork barrier at the 3' end of yeast ribosomal RNA genes.
Replication of the approximately 200 tandem copies of yeast ribosomal RNA genes (rDNA) is known to be initiated within a subset of the repeats, with transcription continuing during the replication process. To examine replication fork movement in this gene cluster, we used a two-dimensional (2D) agarose gel electrophoresis procedure that distinguishes molecules with different branched structures. Replication forks move through most of the rDNA in the same direction in which RNA polymerase I transcribes the 35S rRNA precursor: the 3' end of this transcription unit acts as a barrier to replication forks moving in the direction opposite to RNA polymerase I. The replication fork barrier (RFB) is observed as the accumulation of branched intermediates of specific size. We propose that the act of transcription may influence the movement of replication forks, creating barriers at the 3' ends of actively transcribed genes.
DNA Replication↗
Time of replication of yeast centromeres and telomeres.
The time of replication of centromeres and telomeres of the yeast S. cerevisiae was determined by performing Meselson-Stahl experiments with synchronized cells. The nine centromeres examined become hybrid in density early in S phase, eliminating the possibility that a delay in the replication of centromeres until mitosis is responsible for sister chromatid adherence and proper chromosome segregation at anaphase. The conserved sequence element Y', present at most telomeres, replicates late in S phase, as do the unique sequences adjacent to five specific telomeres. The early and late replication times of these structural elements may be either essential for their proper function or a consequence of some architectural feature of the chromosome.
Centromere↗
Differential regulation of the yeast CDC7 gene during mitosis and meiosis.
The product of the CDC7 gene of Saccharomyces cerevisiae is known to be required in the mitotic cell cycle for the initiation of DNA replication. We show that changes in transcript levels do not account for this stage-specific function, since the steady-state mRNA concentration remains constant at 1 copy per cell throughout the cell cycle. By measuring the cell division capacity of a cdc7::URA3 mutant after loss of a single-copy plasmid containing the CDC7 gene, we show that the CDC7 protein is present in at least 200-fold excess of the amount required for a single cell division. These results appear to exclude periodic transcription or translation as a means by which CDC7 function is regulated. In contrast, the CDC7 protein is known to be dispensable for meiotic S phase, but is required for synaptonemal complex formation and recombination. We found that the CDC7 transcript level does vary during meiosis, reaching a maximum near the time at which recombination occurs. Meiotic spores containing a cdc7 null allele germinate but fail to complete cell division. Apparently the excess CDC7 product present in mitotic cells is physically excluded from the spores (or becomes inactivated) and must be produced de novo after germination. The cdc7-1 allele had previously been shown to confer a reduction in the rate of induced mutation. We show that the cloned wild-type CDC7 gene not only complements this defect, but that when the CDC7 gene is on a multiple copy plasmid, induced mutagenesis is increased. Therefore, in contrast to the excess CDC7 activity for cell division, the level of activity for some error-prone repair process may be normally limiting.
Cell Cycle↗
Copy number and partition of the Saccharomyces cerevisiae 2 micron plasmid controlled by transcription regulators.
The 2 micron plasmid of Saccharomyces cerevisiae is maintained by the action of plasmid-encoded gene products that control copy number and promote equipartition of plasmid copies at cell division. We show that the REP1 and REP2 plasmid-encoded gene products are master regulators that act in concert to autoregulate the level of their own transcripts and to regulate transcript levels of the FLP gene that promotes plasmid copy amplification. REP1 and REP2 are also shown to repress transcription at REP3, the cis-acting site essential for plasmid equipartitioning. We propose a model in which REP3 acts by dislodging transcription apparatuses that otherwise cause plasmid molecules to adhere to the mother nucleus and segregate asymmetrically. On the basis of their ability to generate specific chromatin structures, we also propose that the REP1 and REP2 gene products interact with different specific sequences found iterated in the 2 micron plasmid.
Base Sequence↗
The localization of replication origins on ARS plasmids in S. cerevisiae.
Replication intermediates from the yeast 2 microns plasmid and a recombinant plasmid containing the yeast autonomous replication sequence ARS1 have been analyzed by two-dimensional agarose gel electrophoresis. Plasmid replication proceeds through theta-shaped (Cairns) intermediates, terminating in multiply interlocked catenanes that are resolved during S phase to monomer plasmids. Restriction fragments derived from the Cairns forms contain replication forks and bubbles that behave differently from one another when subjected to high voltage and agarose concentrations. The two-dimensional gel patterns observed for different restriction fragments from these two plasmids indicate that in each plasmid there is a single, specific origin of replication that maps, within the limits of our resolution, to the ARS element. Our results strongly support the long-standing assumption that in Saccharomyces cerevisiae an ARS is an origin of replication.
DNA Replication↗