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     

Rif1 regulates the replication timing domains on the human genome

DNA replication is spatially and temporally regulated during S-phase. DNA replication timing is established in early-G1-phase at a point referred to as timing decision point. However, how the genome-wide replication timing domains are established is unknown. Here, we show that Rif1 (Rap1-interacting-factor-1), originally identified as a telomere-binding factor in yeast, is a critical determinant of the replication timing programme in human cells. Depletion of Rif1 results in specific loss of mid-S replication foci profiles, stimulation of initiation events in early-S-phase and changes in long-range replication timing domain structures. Analyses of replication timing show replication of sequences normally replicating early is delayed, whereas that normally replicating late is advanced, suggesting that replication timing regulation is abrogated in the absence of Rif1. Rif1 tightly binds to nuclear-insoluble structures at late-M-to-early-G1 and regulates chromatin-loop sizes. Furthermore, Rif1 colocalizes specifically with the mid-S replication foci. Thus, Rif1 establishes the mid-S replication domains that are restrained from being activated at early-S-phase. Our results indicate that Rif1 plays crucial roles in determining the replication timing domain structures in human cells through regulating higher-order chromatin architecture.     

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.     

Fiber autoradiography of replicating yeast DNA.

The replication of yeast nuclear DNA was examined by DNA fiber autoradiography. We found that yeast chromosomes contain multiple initiation sites for DNA synthesis. DNA is replicated bidirectionally from these sites at a rate of approx. 0.7 μm/min/replication fork at 24 °C. In these respects DNA replication in this simple eucaryote is similar to DNA replication in mammalian cells.     

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.     

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.     

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.     

A role for yeast and human translesion synthesis DNA polymerases in promoting replication through 3-methyl adenine

3-Methyl adenine (3meA), a minor-groove DNA lesion, presents a strong block to synthesis by replicative DNA polymerases (Pols). To elucidate the means by which replication through this DNA lesion is mediated in eukaryotic cells, here we carry out genetic studies in the yeast Saccharomyces cerevisiae treated with the alkylating agent methyl methanesulfonate. From the studies presented here, we infer that replication through the 3meA lesion in yeast cells can be mediated by the action of three Rad6-Rad18-dependent pathways that include translesion synthesis (TLS) by Pol(eta) or -zeta and an Mms2-Ubc13-Rad5-dependent pathway which presumably operates via template switching. We also express human Pols iota and kappa in yeast cells and show that they too can mediate replication through the 3meA lesion in yeast cells, indicating a high degree of evolutionary conservation of the mechanisms that control TLS in yeast and human cells. We discuss these results in the context of previous observations that have been made for the roles of Pols eta, iota, and kappa in promoting replication through the minor-groove N2-dG adducts.     

A replication enhancer of viral origin increases the mitotic stability of yeast transformants]

It is shown in this paper that a DNA fragment of Hepatitis B virus possessing structural features of yeast replication enhancer increases the mitotic stability of yeast transformants containing hybrid plasmids of episomal and replicative types. The mitotic stability of transformants with plasmid of the replicative type and with the replication enhancer increases only in [cir+] cells. Comparison of primary sequences of HBV DNA of different subtypes revealed that only DNA has unique structural features of the yeast enhancer of replication.     

Apoptosis-like yeast cell death in response to DNA damage and replication defects

In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.     

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.     

DNA Damage Enhanced by the Attenuation of SLD5 Delays Cell Cycle Restoration in Normal Cells but Not in Cancer Cells

SLD5 is a member of the GINS complex composed of PSF1, PSF2, PSF3 and SLD5, playing a critical role in the formation of the DNA replication fork with CDC45 in yeast. Previously, we had isolated a PSF1 orthologue from a murine hematopoietic stem cell DNA library and were then able to identify orthologues of all the other GINS members by the yeast two hybrid approach using PSF1 as the bait. These GINS orthologues may also function in DNA replication in mammalian cells because they form tetrameric complexes as observed in yeast, and gene deletion mutants of both PSF1 and SLD5 result in a lack of epiblast proliferation and early embryonic lethality. However, we found that PSF1 is also involved in chromosomal segregation in M phase, consistent with recent suggestions that homologues of genes associated with DNA replication in lower organisms also regulate cellular events other than DNA replication in mammalian cells. Here we analyzed the function of SLD5 other than DNA replication and found that it is active in DNA damage and repair. Attenuation of SLD5 expression results in marked DNA damage in both normal cells and cancer cells, suggesting that it protects against DNA damage. Attenuation of SLD5 delays the DNA repair response and cell cycle restoration in normal cells but not in cancer cells. These findings suggest that SLD5 might represent a therapeutic target molecule acting at the level of tumor stromal cells rather than the cancerous cells themselves, because development of the tumor microenvironment could be delayed or disrupted by the suppression of its expression in the normal cell types within the tumor.     

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.     

Mechanisms involved in regulating DNA replication origins during the cell cycle and in response to DNA damage

Replication origins in eukaryotic cells never fire more than once in a given S phase. Here, we summarize the role of cyclin-dependent kinases in limiting DNA replication origin usage to once per cell cycle in the budding yeast Saccharomyces cerevisiae. We have examined the role of different cyclins in the phosphorylation and regulation of several replication/regulatory factors including Cdc6, Sic1, ORC and DNA polymerase alpha-primase. In addition to being regulated by the cell cycle machinery, replication origins are also regulated by the genome integrity checkpoint kinases, Mec1 and Rad53. In response to DNA damage or drugs which interfere with the progression of replication forks, the activation of late-firing replication origins is inhibited. There is evidence indicating that the temporal programme of origin firing depends upon the local histone acetylation state. We have attempted to test the possibility that checkpoint regulation of late-origin firing operates through the regulation of the acetylation state. We found that overexpression of the essential histone acetylase, Esal, cannot override checkpoint regulation of origin firing. We have also constructed a temperature-sensitive esa1 mutant. This mutant is unable to resume cell cycle progression after alpha-factor arrest. This can be overcome by overexpression of the G1 cyclin, Cln2, revealing a novel role for Esal in regulating Start.     

Replication and recombination intersect

A bacterial housekeeping function, which requires both recombination and replication enzymes, has been identified that re-establishes inactivated replication forks under normal growth conditions. Some long-tract gene-conversion events initiated by double-strand breaks in yeast and mammalian cells can be attributed to recombination-directed DNA replication. Double-strand break repair in yeast has been shown to require both leading- and lagging-strand DNA synthesis. These observations suggest that the recombination and replication machinery cooperate to maintain genomic integrity.     

Binding properties of replication protein A from human and yeast cells.

Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70, 32, and 14 kDa). We have analyzed the DNA-binding properties of both human and yeast RP-A in order to gain a better understanding of their role(s) in DNA replication. Human RP-A has high affinity for single-stranded DNA and low affinity for RNA and double-stranded DNA. The apparent affinity constant of RP-A for single-stranded DNA is in the range of 10(9) M-1. RP-A has a binding site size of approximately 30 nucleotides and does not bind cooperatively. The binding of RP-A to single-stranded DNA is partially sequence dependent. The affinity of human RP-A for pyrimidines is approximately 50-fold higher than its affinity for purines. The binding properties of yeast RP-A are similar to those of the human protein. Both yeast and human RP-A bind preferentially to the pyrimidine-rich strand of a homologous origin of replication: the ARS307 or the simian virus 40 origin of replication, respectively. This asymmetric binding suggests that RP-A could play a direct role in the process of initiation of DNA replication.     

Temporal order in yeast chromosome replication

Previous work with bacteria has shown that a gene is maximally sensitive to mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine (NG) at the time it is being replicated. NG was used to test for temporal order in the replication of the genome of the unicellular eucaryote, Saccaromyces cerevisiae. Yeast cells growing exponentially were more sensitive to mutagenesis by NG than cells in which DNA synthesis had been inhibited. Further, in a synchronized population of cells, individual genetic markers exhibited maximum sensitivity to mutagenesis at distinct limited intervals within the DNA synthesis period. The peaks of sensitivity are interpreted as reflecting the times of replication of different genes. Since markers for five genes on four different chromosomes showed discrete periods of maximum sensitivity, it is likely that temporal ordering of replication exists for most genes in the yeast genome. These results imply that sites for initiation of DNA replication occur at fairly specific regions along yeast chromosomal DNA molecules, and are activated at predetermined times in the DNA synthesis period.     

Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation

Mcm10 is essential for chromosome replication in eukaryotic cells and was previously thought to link the Mcm2-7 DNA helicase at replication forks to DNA polymerase alpha. Here, we show that yeast Mcm10 interacts preferentially with the fraction of the Mcm2-7 helicase that is loaded in an inactive form at origins of DNA replication, suggesting a role for Mcm10 during the initiation of chromosome replication, but Mcm10 is not a stable component of the replisome subsequently. Studies with budding yeast and human cells indicated that Mcm10 chaperones the catalytic subunit of polymerase alpha and preserves its stability. We used a novel degron allele to inactivate Mcm10 efficiently and this blocked the initiation of chromosome replication without causing degradation of DNA polymerase alpha. Strikingly, the other essential helicase subunits Cdc45 and GINS were still recruited to Mcm2-7 when cells entered S-phase without Mcm10, but origin unwinding was blocked. These findings indicate that Mcm10 is required for a novel step during activation of the Cdc45-MCM-GINS helicase at DNA replication origins.     

Identification of Yeast Factors Involved in the Replication of Mungbean Yellow Mosaic India Virus Using Yeast Temperature-Sensitive Mutants

正Dear Editor,The geminiviruses are small single-stranded plant DNA viruses belonging to the family Geminiviridae, which cause serious diseases in many economically important