Replication fork rate and origin activation during the S phase of Saccharomyces cerevisiae.

When the growth rate of the yeast Saccharomyces cerevisiae is limited with various nitrogen sources, the duration of the S phase is proportional to cell cycle length over a fourfold range of growth rates (C.J. Rivin and W. L. Fangman, 1980, J. Cell Biol. 85:96-107). Molecular parameters of the S phases of these cells were examined by DNA fiber autoradiography. Changes in replication fork rate account completely for the changes in S-phase duration. No changes in origin-to-origin distances were detected. In addition, it was found that while most adjacent replication origins are activated within a few minutes of each other, new activations occur throughout the S phase.

Cell cycle phase expansion in nitrogen-limited cultures of Saccharomyces cerevisiae.

The time and coordination of cell cycle events were examined in the budding yeast Saccharomyces cerevisiae. Whole-cell autoradiographic techniques and time-lapse photography were used to measure the duration of the S, G1, and G2 phases, and the cell cycle positions of "start" and bud emergence, in cells whose growth rates were determined by the source of nitrogen. It was observed that the G1, S, and G2 phases underwent a proportional expansion with increasing cell cycle length, with the S phase occupying the middle half of the cell cycle. In each growth condition, start appeared to correspond to the G1 phase/S phase boundary. Bud emergence did not occur until mid S phase. These results show that the rate of transit through all phases of the cell cycle can vary considerably when cell cycle length changes. When cells growing at different rates were arrested in G1, the following synchronous S phase were of the duration expected from the length of S in each asynchronous population. Cells transferred from a poor nitrogen source to a good one after arrest in G1 went through the subsequent S phase at a rate characteristic of the better medium, indicating that cells are not committed in G1 to an S phase of a particular duration.

Initiation at closely spaced replication origins in a yeast chromosome.

Replication of eukaryotic chromosomes involves initiation at origins spaced an average of 50 to 100 kilobase pairs. In yeast, potential origins can be recognized as autonomous replication sequences (ARSs) that allow maintenance of plasmids. However, there are more ARS elements than active chromosomal origins. The possibility was examined that close spacing of ARSs can lead to inactive origins. Two ARSs located 6.5 kilobase pairs apart can indeed interfere with each other. Replication is initiated from one or the other ARS with equal probability, but rarely (< 5%) from both ARSs on the same DNA molecule.

Cell cycle-dependent establishment of a late replication program.

DNA replication origins in chromosomes of eukaryotes are activated according to a temporal program. In the yeast Saccharomyces cerevisiae, activation of origins in early S phase appears to be a default state. However, cis-acting elements such as telomeres can delay origin activation until late S phase. Site-specific recombination was used to separate origin from telomere in vivo, thereby demonstrating that the signal for late activation is established between mitosis and START in the subsequent G1 phase. Once set, the signal can persist through the next S phase in the absence of the telomere. Establishment of the temporal program and of initiation competence of origins may be coincident events.

Replication dynamics of the yeast genome.

Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae. The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays. Origin activations take place continuously throughout S phase but with most firings near mid-S phase. Rates of replication fork movement vary greatly from region to region in the genome. The two ends of each of the 16 chromosomes are highly correlated in their times of replication. This microarray approach is readily applicable to other organisms, including humans.

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.

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

We have used gene disruptions and nuclease probes to assess the roles of yeast 2 micron plasmid genes in plasmid chromatin organization. The chromatin structure at the replication origin is not dependent on any of the four major open reading frames, A, B, C, or D. While stable plasmid maintenance is known to depend on a cis-acting locus STB and genes B and C, we find that only gene B influences STB chromatin. Other interactions between plasmid gene products and sequences may reflect gene regulation: the chromatin organization at the 5' end of gene A, which codes for a site-specific recombinase, depends on both gene B and gene C. Since disruption of gene C results in an increase in plasmid copy number that is dependent on gene A, we propose that gene C (and probably gene B) control copy number by regulating the level of the gene A recombinase.

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.

Conservative replication of double-stranded RNA in Saccharomyces cerevisiae by displacement of progeny single strands.

Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) molecules, L and M, encapsulated in virus-like particles. After cells are transferred from dense (13C 15N) to light (12C 14N) medium, only two density classes of dsRNA are found, fully light (LL) and fully dense (HH). Cells contain single-stranded copies of both dsRNAs and, at least for L dsRNA, greater than 99% of these single strands are the positive protein-encoding strand. Single-stranded copies of L and M dsRNA accumulate rapidly in cells arrested in the G1 phase. These results parallel previous observations on L dsRNA synthesis and are consistent with a role of the positive single strands as intermediates in dsRNA replication. We propose that new positive strands are displaced from parental molecules and subsequently copied to produce the completely new duplexes.

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.

A test of the transcription model for biased inheritance of yeast mitochondrial DNA.

Two strand-specific origins of replication appear to be required for mammalian mitochondrial DNA (mtDNA) replication. Structural equivalents of these origins are found in the rep sequences of Saccharomyces cerevisiae mtDNA. These striking similarities have contributed to a universal model for the initiation of mtDNA replication in which a primer is created by cleavage of an origin region transcript. Consistent with this model are the properties of deletion mutants of yeast mtDNA ([rho-]) with a high density of reps (HS [rho-]). These mutant mtDNAs are preferentially inherited by the progeny resulting from the mating of HS [rho-] cells with cells containing wild-type mtDNA ([rho+]). This bias is presumed to result from a replication advantage conferred on HS [rho-] mtDNA by the high density of rep sequences acting as origins. To test whether transcription is indeed required for the preferential inheritance of HS [rho-] mtDNA, we deleted the nuclear gene (RPO41) for the mitochondrial RNA polymerase, reducing transcripts by at least 1000-fold. Since [rho-] genomes, but not [rho+] genomes, are stable when RPO41 is deleted, we examined matings between HS [rho-] and neutral [rho-] cells. Neutral [rho-] mtDNAs lack rep sequences and are not preferentially inherited in [rho-] x [rho+] crosses. In HS [rho-] x neutral [rho-] matings, the HS [rho-] mtDNA was preferentially inherited whether both parents were wild type or both were deleted for RPO41. Thus, transcription from the rep promoter does not appear to be necessary for biased inheritance. Our results, and analysis of the literature, suggest that priming by transcription is not a universal mechanism for mtDNA replication initiation.

Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.

Yeast L double-stranded ribonucleic acid is synthesized during the G1 phase but not the S phase of the cell cycle.

The cytoplasm of Saccharomyces cerevisiae contains two major classes of protein-encapsulated double-stranded ribonucleic acids (dsRNA's), L and M. Replication of L and M dsRNA's was examined in cells arrested in the G1 phase by either alpha-factor, a yeast mating pheromone, or the restrictive temperature for a cell cycle mutant (cdc7). [3H]uracil was added during the arrest periods to cells prelabeled with [14C]uracil, and replication was monitored by determining the ratio of 3H/14C for purified dsRNA's. Like mitochondrial deoxyribonucleic acid, both L and M dsRNA's were synthesized in the G1 arrested cells. The replication of L dsRNA was also examined during the S phase, using cells synchronized in two different ways. Cells containing the cdc7 mutation, treated sequentially with alpha-factor and then the restrictive temperature, enter a synchronous S phase when transferred to permissive temperature. When cells entered the S phase, synthesis of L dsRNA ceased, and little or no synthesis was detected throughout the S phase. Synthesis of L dsRNA was also observed in G1 phase cells isolated from asynchronous cultures by velocity centrifugation. Again, synthesis ceased when cells entered the S phase. These results indicate that L dsRNA replication is under cell cycle control. The control differs from that of mitochondrial deoxyribonucleic acid, which replicates in all phases of the cell cycle, and from that of 2-micron DNA, a multiple-copy plasmid whose replication is confined to the S phase.

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.

Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae.

The product of the CDC7 gene of Saccharomyces cerevisiae appears to have multiple roles in cellular physiology. It is required for the initiation of mitotic DNA synthesis. While it is not required for the initiation of meiotic DNA replication, it is necessary for genetic recombination during meiosis and for the formation of ascospores. It has also been implicated in an error-prone DNA repair pathway. Plasmids capable of complementing temperature-sensitive cdc7 mutations were isolated from libraries of yeast genomic DNA in the multicopy plasmid vectors YRp7 and YEp24. The complementing activity was localized within a 3.0-kilobase genomic DNA fragment. Genetic studies that included integration of the genomic insert at or near the CDC7 locus and marker rescue of four cdc7 alleles proved that the cloned fragment contains the yeast chromosomal CDC7 gene. The RNA transcript of CDC7 is about 1,700 nucleotides. Analysis of the nucleotide sequence of a 2.1-kilobase region of the cloned fragment revealed the presence of an open reading frame of 1,521 nucleotides that is presumed to encode the CDC7 protein. Depending on which of two possible ATG codons initiates translation, the calculated size of the CDC7 protein is 58.2 or 56 kilodaltons. Comparison of the predicted amino acid sequence of the CDC7 gene product with other known protein sequences suggests that CDC7 encodes a protein kinase.

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.

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.

Active role of a human genomic insert in replication of a yeast artificial chromosome.

Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryotic DNA. The manner by which large pieces of foreign DNA are assimilated by yeast cells into a functional chromosome is poorly understood, as is the reason why some of them are stably maintained and some are not. We examined the replication of a stable YAC containing a 240-kb insert of DNA from the human T-cell receptor beta locus. The human insert contains multiple sites that serve as origins of replication. The activity of these origins appears to require the yeast ARS consensus sequence and, as with yeast origins, additional flanking sequences. In addition, the origins in the human insert exhibit a spacing, a range of activation efficiencies, and a variation in times of activation during S phase similar to those found for normal yeast chromosomes. We propose that an appropriate combination of replication origin density, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.

Ribosomal DNA replication fork barrier and HOT1 recombination hot spot: shared sequences but independent activities.

In the ribosomal DNA of Saccharomyces cerevisiae, sequences in the nontranscribed spacer 3' of the 35S ribosomal RNA gene are important to the polar arrest of replication forks at a site called the replication fork barrier (RFB) and also to the cis-acting, mitotic hyperrecombination site called HOT1. We have found that the RFB and HOT1 activity share some but not all of their essential sequences. Many of the mutations that reduce HOT1 recombination also decrease or eliminate fork arrest at one of two closely spaced RFB sites, RFB1 and RFB2. A simple model for the juxtaposition of RFB and HOT1 sequences is that the breakage of strands in replication forks arrested at RFB stimulates recombination. Contrary to this model, we show here that HOT1-stimulated recombination does not require the arrest of forks at the RFB. Therefore, while HOT1 activity is independent of replication fork arrest, HOT1 and RFB require some common sequences, suggesting the existence of a common trans-acting factor(s).

Thymidine utilization by tut mutants and facile cloning of mutant alleles by plasmid conversion in S. cerevisiae.

Plasmid pJM81 contains a Herpes simplex virus thymidine kinase (TK) gene that is expressed in yeast. Cells containing the plasmid utilize thymidine (TdR) and the analogue 5-bromodeoxyuridine (BUdR) for specific incorporation into DNA. TdR auxotrophs, harboring plasmid pJM81 and a mutation in the yeast gene TMP1 require high concentrations of TdR (300 micrograms/ml) to support normal growth rates and the wild-type mitochondrial genome (rho+) cannot be maintained. We have identified a yeast gene, TUT1, in which recessive mutations allow efficient utilization of lower concentrations of TdR. Strains containing the mutations tmp1 and tut1, as well as plasmid pJM81, form colonies at 2 micrograms/ml TdR, grow at nearly normal rates and maintain the rho+ genome at 50 micrograms/ml TdR. These strains can be used to radiolabel DNA specifically and to synchronize DNA replication by TdR starvation. In addition, the substitution of BUdR for TdR allows the selective killing of DNA-synthesizing cells by 310-nm irradiation and allows the separation of replicated and unreplicated forms of DNA by CsCl equilibrium density banding. We also describe a unique, generally applicable system for cloning mutant alleles that exploits the fact that Tk+ yeast cells are sensitive to 5-fluorodeoxyuridine (FUdR) and that gene conversions can occur between a yeast chromosome and a TK-containing plasmid.