The replication behavior of Saccharomyces cerevisiae DNA in human cells

We studied the replication of random genomic DNA fragments from Saccharomyces cerevisiae in a long-term assay in human cells. Plasmids carrying large yeast DNA fragments were able to replicate autonomously in human cells. Efficiency of replication of yeast DNA fragments was comparable to that of similarly sized human DNA fragments and better than that of bacterial DNA. This result suggests that yeast genomic DNA contains sequence information needed for replication in human cells. To examine whether DNA replication in human cells would initiate specifically at a yeast origin of replication, we monitored initiation on a plasmid containing the yeast 2-micron autonomously replicating sequence (ARS) in yeast and human cells. We found that while replication initiates at the 2-micron ARS in yeast, it does not preferentially initiate at the ARS in human cells. This result suggests that the sequences that direct site specific replication initiation in yeast do not function in the same way in human cells, which initiate replication at a broader range of sequences.by J.A. Huberman     

Checkpoint independence of most DNA replication origins in fission yeast

Background

In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2).

Results

Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells.

Conclusion

The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.     

Origin plasticity during budding yeast DNA replication in vitro

The separation of DNA replication origin licensing and activation in the cell cycle is essential for genome stability across generations in eukaryotic cells. Pre‐replicative complexes (pre‐RCs) license origins by loading Mcm2‐7 complexes in inactive form around DNA. During origin firing in S phase, replisomes assemble around the activated Mcm2‐7 DNA helicase. Budding yeast pre‐RCs have previously been reconstituted in vitro with purified proteins. Here, we show that reconstituted pre‐RCs support replication of plasmid DNA in yeast cell extracts in a reaction that exhibits hallmarks of cellular replication initiation. Plasmid replication in vitro results in the generation of covalently closed circular daughter molecules, indicating that the system recapitulates the initiation, elongation, and termination stages of DNA replication. Unexpectedly, yeast origin DNA is not strictly required for DNA replication in vitro, as heterologous DNA sequences could support replication of plasmid molecules. Our findings support the notion that epigenetic mechanisms are important for determining replication origin sites in budding yeast, highlighting mechanistic principles of replication origin specification that are common among eukaryotes.     

Study of animal viruses in yeast

Yeast is often considered to be a model eukaryotic organism, in a manner analogous to E. coli as a model prokaryotic organism. Yeast has been extensively characterized and the genomes completely sequenced. Despite the small genome size, yeast displays most of features of higher eukaryotes. The facts that most of cellular machinery is conserved among different eukaryotes and that the powerful technologies of genetics and molecular biology are available have made yeast model eukaryotic cells in biological and biomedical sciences including virology. Cumulative data indicate that yeast can be a host for animal viruses. I briefly describe yeast gene expression and review viral replication in yeast. Great discovery include complete replication of animal viruses and production of virus-like particle vaccines in yeast. Current studies on yeast focus on identification of host factors and machinery used for viral replication. The studies are based on traditional yeast genetics and genome-wide identification using a complete set of yeast deletion strains.     

Association of Fission Yeast Orp1 and Mcm6 Proteins with Chromosomal Replication Origins

We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by the nda1(+)/cdc19(+) and mis5(+) genes, respectively, were associated with chromatin DNA only during the G(1) and S phases. Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G(1) and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 and ars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G(1) and S phases. Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication.     

Schizosaccharomyces pombe Noc3 is essential for ribosome biogenesis and cell division but not DNA replication

The initiation of eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at chromosomal origins of DNA replication. Pre-RC assembly requires the essential DNA replication proteins ORC, Cdc6, and Cdt1 to load the MCM DNA helicase onto chromatin. Saccharomyces cerevisiae Noc3 (ScNoc3), an evolutionarily conserved protein originally implicated in 60S ribosomal subunit trafficking, has been proposed to be an essential regulator of DNA replication that plays a direct role during pre-RC formation in budding yeast. We have cloned Schizosaccharomyces pombe noc3(+) (Spnoc3(+)), the S. pombe homolog of the budding yeast ScNOC3 gene, and functionally characterized the requirement for the SpNoc3 protein during ribosome biogenesis, cell cycle progression, and DNA replication in fission yeast. We showed that fission yeast SpNoc3 is a functional homolog of budding yeast ScNoc3 that is essential for cell viability and ribosome biogenesis. We also showed that SpNoc3 is required for the normal completion of cell division in fission yeast. However, in contrast to the proposal that ScNoc3 plays an essential role during DNA replication in budding yeast, we demonstrated that fission yeast cells do enter and complete S phase in the absence of SpNoc3, suggesting that SpNoc3 is not essential for DNA replication in fission yeast.     

Yeast DNA replication in vitro: initiation and elongation events mimic in vivo processes

An enzyme system prepared from Saccharomyces cerevisiae carries out the replication of exogenous yeast plasmid DNA. Replication in vitro mimics that in vivo in that DNA synthesis in extracts of strain cdc8, a temperature-sensitive DNA replication mutant, is thermolabile relative to the wild-type, and in that aphidicolin inhibits replication in vitro. Furthermore, only plasmids containing a functional yeast replicator, ARS, initiate replication at a specific site in vitro. Analysis of replicative intermediates shows that plasmid YRp7, which contains the chromosomal replicator ARS1, initiates bidirectional replication in a 100 bp region within the sequence required for autonomous replication in vivo. Plasmids containing ARS2, another chromosomal replicator, and the ARS region of the endogenous yeast plasmid 2 microns circle give similar results, suggesting that ARS sequences are specific origins of chromosomal replication. Used in conjunction with deletion mapping, the in vitro system allows definition of the minimal sequences required for the initiation of replication.     

Identification of Novel Host Factors via Conserved Domain Search: Cns1 Cochaperone Is a Novel Restriction Factor of Tombusvirus Replication in Yeast

A large number of host-encoded proteins affect the replication of plus-stranded RNA viruses by acting as susceptibility factors. Many other cellular proteins are known to function as restriction factors of viral infections. Previous studies with tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the inhibitory function of TPR (tetratricopeptide repeat) domain-containing cyclophilins, which are members of the large family of host prolyl isomerases, in TBSV replication. In this paper, we tested additional TPR-containing yeast proteins in a cell-free TBSV replication assay and identified the Cns1p cochaperone for heat shock protein 70 (Hsp70) and Hsp90 chaperones as a strong inhibitor of TBSV replication. Cns1p interacted with the viral replication proteins and inhibited the assembly of the viral replicase complex and viral RNA synthesis in vitro. Overexpression of Cns1p inhibited TBSV replication in yeast. The use of a temperature-sensitive (TS) mutant of Cns1p in yeast revealed that at a semipermissive temperature, TS Cns1p could not inhibit TBSV replication. Interestingly, Cns1p and the TPR-containing Cpr7p cyclophilin have similar inhibitory functions during TBSV replication, although some of the details of their viral restriction mechanisms are different. Our observations indicate that TPR-containing cellular proteins could act as virus restriction factors.     

Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains

Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK(+) yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8-9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 'early' origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.     

Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA.

Transformation studies with Saccharomyces cerevisiae (bakers' yeast) have identified DNA sequences which permit extrachromosomal maintenance of recombinant DNA plasmids in transformed cells. It has been hypothesized that such sequences (called ARS for autonomously replicating sequence) serve as initiation sites for DNA replication in recombinant DNA plasmids and that they represent the normal sites for initiation of replication in yeast chromosomal DNA. We have constructed a novel plasmid called TRP1 R1 Circle which consists solely of 1,453 base pairs of yeast chromosomal DNA. TRP1 RI Circle contains both the TRP1 gene and a sequence called ARS1. This plasmid is found in 100 to 200 copies per cell and is relatively stable during both mitotic and meiotic cell cycles. Replication of TRP1 RI Circle requires the products of the same genes (CDC28, CDC4, CDC7, and CDC8) required for replication of chromosomaL DNA. Like chromosomal DNA, its replication does not occur in cells arrested in the B1 phase of the cell cycle by incubation with the yeast pheromone alpha-factor. In addition, TRP1 RI Circle DNA is organized into nucleosomes whose size and spacing are indistinguishable from that of bulk yeast chromatin. These results indicate that TRP1 RI Circle has the replicative and structural properties expected for an origin of replication from yeast chromosomal DNA. Thus, this plasmid is a suitable model for further studies of yeast DNA replication in both cells and cell-free extracts.     

Genetic methods for characterizing the cis-acting components of yeast DNA replication origins.

Small circular plasmids containing replication origins and, in some cases, centromeres, can replicate autonomously in the nuclei of all tested yeast species. Because this autonomous replication is dependent on the replication origin within the plasmid, measurements of the efficiency of autonomous replication (by the methods summarized here) permit evaluation of the effects of mutations on origin function. Although alternative methods are available for genetic characterization of replication origins in other organisms, the simplicity of the autonomous replication assay in yeasts has permitted development of the deepest understanding to date of eukaryotic replication origin structure. This information has come primarily from studies with Saccharomyces cerevisiae. However, there are many other yeast species, each with its own variety of replication origins. Use of the methods summarized here to characterize origins in other yeast species is likely to provide additional insights into eukaryotic replication origin structure.     

A structured model and likelihood approach to estimate yeast prion propagon replication rates and their asymmetric transmission

Prion proteins cause a variety of fatal neurodegenerative diseases in mammals but are generally harmless to Baker’s yeast (Saccharomyces cerevisiae). This makes yeast an ideal model organism for investigating the protein dynamics associated with these diseases. The rate of disease onset is related to both the replication and transmission kinetics of propagons, the transmissible agents of prion diseases. Determining the kinetic parameters of propagon replication in yeast is complicated because the number of propagons in an individual cell depends on the intracellular replication dynamics and the asymmetric division of yeast cells within a growing yeast cell colony. We present a structured population model describing the distribution and replication of prion propagons in an actively dividing population of yeast cells. We then develop a likelihood approach for estimating the propagon replication rate and their transmission bias during cell division. We first demonstrate our ability to correctly recover known kinetic parameters from simulated data, then we apply our likelihood approach to estimate the kinetic parameters for six yeast prion variants using propagon recovery data. We find that, under our modeling framework, all variants are best described by a model with an asymmetric transmission bias. This demonstrates the strength of our framework over previous formulations assuming equal partitioning of intracellular constituents during cell division.     

Processing of telomeric DNA ends requires the passage of a replication fork.

During telomere replication in yeast, chromosome ends acquire a long single-stranded extension of the strand making the 3' end. Previous work showed that these 3' tails are generated late in S-phase, when conventional replication is virtually complete. In addition, the extensions were also observed in cells that lacked telomerase. Therefore, a model was proposed that predicted an activity that recessed the 5' ends at yeast telomeres after conventional replication was complete. Here, we demonstrate that this processing activity is dependent on the passage of a replication fork through yeast telomeres. A non-replicating linear plasmid with telomeres at each end does not acquire single-stranded extensions, while an identical construct containing an origin of replication does. Thus, the processing activity could be associated with the enzymes at the replication fork itself, or the passage of the fork through the telomeric sequences allows a transient access for the activity to the telomeres. We therefore propose that there is a mechanistic link between the conventional replication machinery and telomere maintenance.     

ARS replication during the yeast S phase

A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARS1. Isotope density transfer experiments show that each plasmid molecule replicates once each S phase, with initiation depending on two genetically defined steps required for nuclear DNA replication. A density transfer experiment with synchronized cells demonstrates that the ARS1 plasmid population replicates early in the S phase. The sequences adjacent to ARS1 on chromosome IV also initiate replication early, suggesting that the ARS1 plasmid contains information which determines its time of replication. The times of replication for two other yeast chromosome sequences, ARS2 and a sequence referred to as 1OZ, indicate that the temporal order of replication is ARS1 leads to ARS2 leads to 1OZ. These experiments show directly that specific chromosome regions replicate at specific times during the yeast S phase. If ARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins.     

Structural properties of replication origins in yeast DNA sequences

Sequence-dependent DNA flexibility is an important structural property originating from the DNA 3D structure. In this paper, we investigate the DNA flexibility of the budding yeast (S. Cerevisiae) replication origins on a genome-wide scale using flexibility parameters from two different models, the trinucleotide and the tetranucleotide models. Based on analyzing average flexibility profiles of 270 replication origins, we find that yeast replication origins are significantly rigid compared with their surrounding genomic regions. To further understand the highly distinctive property of replication origins, we compare the flexibility patterns between yeast replication origins and promoters, and find that they both contain significantly rigid DNAs. Our results suggest that DNA flexibility is an important factor that helps proteins recognize and bind the target sites in order to initiate DNA replication. Inspired by the role of the rigid region in promoters, we speculate that the rigid replication origins may facilitate binding of proteins, including the origin recognition complex (ORC), Cdc6, Cdt1 and the MCM2-7 complex.     

Sequences that promote formation of catenated intertwines during termination of DNA replication.

The normal sequence at which SV40 DNA replication terminates (TER) is unusual in that it promotes formation of catenated intertwines when two converging replication forks enter to complete replication (Weaver et al., 1985). Here we show that yeast centromeric sequences also exhibit this phenomenon. CEN3 caused accumulation of late replicating intermediates and catenated dimers in plasmids replicating in mammalian cells, but only when it was located in the termination region (180 degrees from ori), and only when cells were subjected to hypertonic shock to reduce topoisomerase II activity. Therefore, formation of catenated intertwines during termination of DNA replication was sequence dependent, suggesting that topoisomerase II acts behind replication forks in the termination region to remove intertwines generated by unwinding DNA rather than acting after replication is completed and catenates are formed. Under normal physiological conditions, CEN3 did not promote formation of catenated dimers in either mammalian or yeast cells. Therefore, CEN does not maintain association of sister chromatids during mitosis in yeast by introducing stable catenated intertwines during replication.     

Overexpression of multisubunit replication factors in yeast.

Facile genetic and biochemical manipulation coupled with rapid cell growth and low cost of growth media has established the yeast Saccharomyces cerevisiae as a versatile workhorse. This article describes the use of yeast expression systems for the overproduction of complex multipolypeptide replication factors. The regulated overexpression of these factors in yeast provides for a readily accessible and inexpensive source of these factors in large quantities. The methodology is illustrated with the five-subunit replication factor C. Whole-cell extracts are prepared by blending yeast cells with glass beads or frozen yeast with dry ice. Procedures are described that maximize the yield of these factors while minimizing proteolytic degradation.     

DNA replication: Pif1 pulls the plug on stalled replication forks

The conserved PIF helicase family appears to function in replication to ensure termination and passage through regions that slow or arrest replication fork movement. Findings in fission yeast extend evidence from budding yeast, and argue for universal mechanisms that ensure replication integrity.     

Engineered retargeting of viral RNA replication complexes to an alternative intracellular membrane

Positive-strand RNA virus replication complexes are universally associated with intracellular membranes, although different viruses use membranes derived from diverse and sometimes multiple organelles. We investigated whether unique intracellular membranes are required for viral RNA replication complex formation and function in yeast by retargeting protein A, the Flock House virus (FHV) RNA-dependent RNA polymerase. Protein A, the only viral protein required for FHV RNA replication, targets and anchors replication complexes to outer mitochondrial membranes in part via an N-proximal sequence that contains a transmembrane domain. We replaced the FHV protein A mitochondrial outer membrane-targeting sequence with the N-terminal endoplasmic reticulum (ER)-targeting sequence from the yeast NADP cytochrome P450 oxidoreductase or inverted C-terminal ER-targeting sequences from the hepatitis C virus NS5B polymerase or the yeast t-SNARE Ufe1p. Confocal immunofluorescence microscopy confirmed that protein A chimeras retargeted to the ER. FHV subgenomic and genomic RNA accumulation in yeast expressing ER-targeted protein A increased 2- to 13-fold over that in yeast expressing wild-type protein A, despite similar protein A levels. Density gradient flotation assays demonstrated that ER-targeted protein A remained membrane associated, and in vitro RNA-dependent RNA polymerase assays demonstrated an eightfold increase in the in vitro RNA synthesis activity of the ER-targeted FHV RNA replication complexes. Electron microscopy showed a change in the intracellular membrane alterations from a clustered mitochondrial distribution with wild-type protein A to the formation of perinuclear layers with ER-targeted protein A. We conclude that specific intracellular membranes are not required for FHV RNA replication complex formation and function.     

Normal Human Telomeres Are Not Late Replicating

Telomeres in yeast are late replicating. Genes placed next to telomeres in yeast can be repressed (telomere positional effects), leading to the hypothesis that telomeres may be heterochromatic and may control the expression of subtelomeric genes. In addition, yeast telomeres are processed to have a transient long overhang at the end of S phase. The applicability of the yeast data to human biology was examined by determining the timing of telomere replication and processing in normal human diploid fibroblasts. Telomeres were purified from synchronized cells that had been labeled with 5-bromodeoxyuridine (BrdU) at hourly intervals, and the fraction of labeled telomeres was analyzed by retrieval with anti-BrdU antibodies. We determined that normal human telomeres replicate throughout S phase rather than being very late replicating. Furthermore, the overall timing of replication was unaffected by telomere length in young versus old cells or cells whose telomeres had been elongated following transfection with the catalytic subunit of telomerase. Finally, the asymmetry in the length of the G-rich overhang in daughter telomeres produced by leading versus lagging strand synthesis was shown to be established within 1 h of telomere replication, indicating there is no significant delay between synthesis and the processing events that contribute to the establishment of asymmetric overhangs. Therefore, the timings of replication and processing of human telomeres are very different from those of yeast.