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PubMed2001

Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae.

Cdc45, which binds to the minichromosomal maintenance (Mcm) proteins, has a pivotal role in the initiation and elongation steps of chromosomal DNA replication in eukaryotes. Here we show that throughout the cell cycle in Saccharomyces cerevisiae, Cdc45 forms a complex with a novel factor, Sld3. Consistently, Sld3 and Cdc45 associate simultaneously with replication origins in the chromatin immunoprecipitation assay: both proteins associate with early-firing origins in G(1) phase and with late-firing origins in late S phase. Moreover, the origin associations of Sld3 and Cdc45 are mutually dependent. The temperature-sensitive sld3 mutation confers a defect in DNA replication at the restrictive temperature and reduces an interaction not only between Sld3 and Cdc45, but also between Cdc45 and Mcm2. These results suggest that the Sld3-Cdc45 complex associates with replication origins through Mcm proteins. At the restrictive temperature in sld3-5 cells, replication factor A, a single-strand DNA binding protein, does not associate with origins. Therefore, the origin association of Sld3-Cdc45 complex is prerequisite for origin unwinding in the initiation of DNA replication.

Carrier Proteins↗
PubMed2004

Embryonic expression of pre-initiation DNA replication factors in Xenopus laevis.

We examined the expression of various DNA replication factors, including: cdc45, the factors of the GINS heterotetramer (Sld5, Psf1, Psf2, Psf3), and PCNA, in Xenopus laevis during embryonic development via whole mount in situ hybridization. For the most part, these factors were expressed in similar patterns, with some subtle variations, throughout development within the anterior CNS, pharyngeal arches, and various placodes. More significant variations were also observed, including expression of only Psf1 and Psf2 in the pronephros and unique Psf2 expression in the somitic mesoderm. Overall, these results suggest that common regulatory mechanisms are involved in the transcriptional deployment of these factors. Significantly, expression of these factors does not directly coincide with elevated patterns of DNA replication, suggesting that different replication factors are utilized in different developmental regions.

Animals↗
PubMed2006

GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks.

The components of the replisome that preserve genomic stability by controlling the progression of eukaryotic DNA replication forks are poorly understood. Here, we show that the GINS (go ichi ni san) complex allows the MCM (minichromosome maintenance) helicase to interact with key regulatory proteins in large replisome progression complexes (RPCs) that are assembled during initiation and disassembled at the end of S phase. RPC components include the essential initiation and elongation factor, Cdc45, the checkpoint mediator Mrc1, the Tof1-Csm3 complex that allows replication forks to pause at protein-DNA barriers, the histone chaperone FACT (facilitates chromatin transcription) and Ctf4, which helps to establish sister chromatid cohesion. RPCs also interact with Mcm10 and topoisomerase I. During initiation, GINS is essential for a specific subset of RPC proteins to interact with MCM. GINS is also important for the normal progression of DNA replication forks, and we show that it is required after initiation to maintain the association between MCM and Cdc45 within RPCs.

Cell Cycle Proteins↗
PubMed1991

Growth and DNA replication in rabbit blastocysts.

DNA content and DNA polymerase activity were measured on rabbit blastocysts removed from the uterus at 24-hr intervals over the period of days 4-7 postcoitum (pc). Median DNA content increased 53 times over the 72-hr period, from 25.3 ng on day 4 to 1,360 ng on day 7. Median DNA polymerase activity (fmole of radiolabeled nucleotide incorporated in 30 min at 37 degrees C) increased 393-fold from day 4 to day 7: 32.8 to 12,900. These embryos also increased in surface area and volume by 334-fold and 6,078-fold, respectively. Litters containing individuals with high DNA content also tended to have similar individuals with high DNA polymerase activity. Therefore, DNA polymerase activity may be a useful measure of the potential for the next cell division. A large amount of variation existed between blastocysts in all parameters measured. An analysis of variance, conducted to partition variation between litters and within litters, determined that within-litter variation was actually greater than that between litters, resulting in intraclass correlation coefficients less than 0.5. There was also a positive regression of DNA content and DNA polymerase activity on surface area in 6- and 7-day-old blastocysts after eliminating variation attributable to litters. The developmental pattern of DNA polymerase activity in the rabbit may be quantitatively different from that described in the mouse. The pattern in mammals is very different from that described in several nonmammalian species.

Animals↗
PubMed1981

Characterization of the DNA-dependent GTPase activity of T4 gene 41 protein, an essential component of the T4 bacteriophage DNA replication apparatus.

The product specified by T4 bacteriophage gene 41 is known from genetic analyses to be essential for phage DNA replication in vivo. Correspondingly, the purified gene 41 protein is an essential component of an efficient in vitro DNA replication system reconstructed from seven purified T4 replication proteins; it is required both for the synthesis of short RNA primers (in conjunction with the T4 gene 61 protein) and for the rapid unwinding of the double-helical DNA template at a replication fork. The purified gene 41 protein exhibits a DNA-dependent GTPase (and ATPase) activity. In this report, we have used this associated GTPase activity as a biochemical probe for the analysis of the interactions between DNA and the 41 protein. Our results suggest that, upon binding GTP, the 41 protein monomer is induced to form a dimer, which can them form a tight complex with single-stranded DNA. Driven by the repeated hydrolysis of GTP molecules, the 41 protein dimer appears to run rapidly along the bound DNA chain. Studies with the synthetic GTP analogue, GTP gamma S, suggest that GTP hydrolysis is required for this 41 protein movement, but that it is not essential for the function of the 41 protein in RNA primer synthesis. In sum, our observations suggest that a 41 protein dimer runs along the lagging strand template at a DNA replication fork; from this position, it functions as a DNA helicase and simultaneously interacts with the T4 gene 61 protein to make the pentaribonucleotide primers which initiate Okazaki pieces at specific primer initiation sites.

DNA Replication↗
PubMed2006

Chromosomal DNA replication in a soluble cell-free system derived from Xenopus eggs.

Cytoplasmic egg extracts from the frog Xenopus laevis represent a powerful cell-free system to study eukaryotic chromosomal DNA replication. In the classical approach, sperm chromatin is added to unfractionated egg cytoplasm, leading to the assembly of transport-competent nuclei that undergo a single, complete round of DNA replication. The need for nuclei in this system has been circumvented. Sperm chromatin or plasmid DNA is first incubated with clarified egg cytoplasm to form chromatin-bound prereplication complexes. Subsequently, a highly concentrated nucleoplasmic extract is added that stimulates initiation from these prereplication complexes, and a single complete round of chromosomal DNA replication ensues. This review describes the preparation of the cytosolic and nucleoplasmic extracts, as well as their use in DNA replication, origin unwinding, and chromatin isolation assays.

Animals↗
PubMed1997

The dual effect of mimosine on DNA replication.

The plant amino acid, mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells, but the mechanism by which this inhibition is achieved is unknown. The drug has been proposed either to inhibit initiation at origins of replication or to inhibit chain elongation by lowering nucleotide pool levels. In an attempt to determine which mode of action is correct, we have analyzed its effects on SV40 DNA replication. Using a two-dimensional gel replicon mapping technique, we show that mimosine completely inhibits incorporation of [3H]thymidine into viral DNA, but only after approximately 4 h. Qualitative analysis of replication intermediates during this interval suggests that the drug partially inhibits both initiation and elongation, and pulse-chase experiments support this contention. The drug has no effect when added directly to an SV40 in vitro replication extract. However, extracts prepared from cells pretreated with mimosine are compromised in their ability to support replication in vitro in the presence of a full complement of nucleotides. Thus, although mimosine may alter nucleotide pool levels in vivo, it also appears to affect one or more essential replication proteins.

Animals↗
PubMed1994

RecA, Tus protein and constitutive stable DNA replication in Escherichia coli rnhA mutants.

Constitutive stable DNA replication (cSDR), which uniquely occurs in Escherichia coli rnhA mutants deficient in ribonuclease HI activity, requires RecA function. The recA428 mutation, which inactivates the recombinase activity but imparts a constitutive coprotease activity, blocks cSDR in rnhA mutants. The result indicates that the recombinase activity of RecA, which promotes homologous pairing and strand exchange, is essential for cSDR. Despite the requirement for RecA recombinase activity, mutations in recB, recD, recJ, ruvA and ruvC neither inhibit nor stimulate cSDR. It was proposed that the property of RecA essential for homologous pairing and strand exchange is uniquely required for initiation of cSDR in rnhA mutants without involving the homologous recombination process. The possibility that RecA protein is necessary to counteract the action of Tus protein, a contra-helicase which stalls replication forks in the ter region of the chromosome, was ruled out because introduction of the tus::kan mutation, which inactivates Tus protein, did not alleviate the RecA requirement for cSDR.

Bacterial Proteins↗
PubMed1977

Effects of mercuric chloride on synchronized Chinese hamster ovary cells: survival and DNA replication.

Chinese hamster ovary (CHO) cells in vitro were treated with HgCl2 at various stages in the cell cycle and the effects of this chemical on cell survival, DNA replication, and cell division were observed. In terms of survival the early G1 cells were the most sensitive to treatment, followed by late G1 and early S, while mid S and late S-G2 treated cells were the least sensitive. Treatment with HgCl2 also resulted in reduced rates of DNA replication and delays in cell division. The early G1 treated cells showed substantially reduced rates of DNA replication followed by 4--5 h division delay. The early S and late S-G2 treated cells had some reduction in their rates of DNA replication followed by corresponding division delay of 2.5 h in the early S treated cells and 1 h in the late S-G2 treated cells.

Animals↗
PubMed1997

Herpes simplex virus DNA replication.

The Herpesviridae comprise a large class of animal viruses of considerable public health importance. Of the Herpesviridae, replication of herpes simplex virustype-1 (HSV-1) has been the most extensively studied. The linear 152-kbp HSV-1 genome contains three origins of DNA replication and approximately 75 open-reading frames. Of these frames, seven encode proteins that are required for originspecific DNA replication. These proteins include a processive heterodimeric DNA polymerase, a single-strand DNA-binding protein, a heterotrimeric primosome with 5'-3' DNA helicase and primase activities, and an origin-binding protein with 3'-5' DNA helicase activity. HSV-1 also encodes a set of enzymes involved in nucleotide metabolism that are not required for viral replication in cultured cells. These enzymes include a deoxyuridine triphosphatase, a ribonucleotide reductase, a thymidine kinase, an alkaline endo-exonuclease, and a uracil-DNA glycosylase. Host enzymes, notably DNA polymerase alpha-primase, DNA ligase I, and topoisomerase II, are probably also required. Following circularization of the linear viral genome, DNA replication very likely proceeds in two phases: an initial phase of theta replication, initiated at one or more of the origins, followed by a rolling-circle mode of replication. The latter generates concatemers that are cleaved and packaged into infectious viral particles. The rolling-circle phase of HSV-1 DNA replication has been reconstituted in vitro by a complex containing several of the HSV-1 encoded DNA replication enzymes. Reconstitution of the theta phase has thus far eluded workers in the field and remains a challenge for the future.

Animals↗
PubMed1997

Studies with artificial extrachromosomal elements in trypanosomatids: could specificity in the initiation of DNA replication be linked to that in transcription?

Historically, artificial replicons have served as useful models for the definition of regulatory elements involved in chromosomal replication and transmission in yeast and DNA replication in bacteria. Here, Pradeep Patnaik examines what we have learnt so far from the replicative behaviour of various artificial extrachromosomal elements available for trypanosomatids. He highlights the involvement of transcription regulatory elements in virtually every eukaryotic origin of replication analysed in detail and, by drawing upon the extensive literature supporting a close association between DNA replication and transcription, he speculates that the nature and organization of origins of replication on a chromosome also may hold clues to the manner by which an organism regulates gene expression.

Journal Article↗
PubMed2005

The ribonucleotide reductase subunit M2B subcellular localization and functional importance for DNA replication in physiological growth of KB cells.

Ribonucleoside diphosphate reductase (EC 1.17.4.1) (RR) is a potential target for antineoplastic agents due to its crucial role in DNA replication and repair. The expression and activity of RR subunits are highly regulated to maintain an optimal dNTP pool, which is required to maintain genetic fidelity. The human RR small subunit M2B (p53R2) is thought to contribute to DNA repair in response to DNA damage. However, it is not clear whether M2B is involved in providing dNTPs for DNA replication under physiological growth conditions. Serum starvation synchronized studies showed that a rapid increase of M2B was associated with cyclin E, which is responsible for regulation of G(1)/S-phase transition. A living cell sorting study that used KB cells in normal growth, further confirmed that M2B increased to maximum levels at the G(1)/S-phase transition, and decreased with DNA synthesis. Confocal studies revealed that M2B redistributed from the cytoplasm to the nucleus earlier than hRRM2 in response to DNA replication. Nuclear accumulation of M2B is associated with dynamic changes in dNTP at early periods of serum addition. By using M2B-shRNA expression vectors, inhibition of M2B may result in growth retardation in KB cells. We conclude that M2B may translocate from the cytoplasm into the nucleus and allow dNTPs to initiate DNA synthesis in KB cells under physiological conditions. Thus, our findings suggested that M2B might play an important role for initiating DNA replication of KB cells in normal growth.

Cell Cycle Proteins↗
PubMed2004

DNA replication initiates at domains overlapping with nuclear matrix attachment regions in the xenopus and mouse c-myc promoter.

Only a very few origins have been mapped in different multicellular organisms, and they do not share detectable consensus sequence elements. Moreover, it is not clear if origins are localized at similar positions in the corresponding locus in genomes of different organisms. Here, we have mapped DNA replication origins in the c-myc locus both in Xenopus and mouse, allowing a comparison of the corresponding sites in three different animal species (Xenopus, mouse, human). An origin of DNA replication is present in the three homologous c-myc loci. In Xenopus, a main DNA replication origin was located 3 kilobases (kb) upstream of the active c-myc promoter, whereas, in mouse, we detected an origin 1 kb upstream of the promoter, as previously mapped in human c-myc. We also identified a nuclear matrix attachment region in both Xenopus and mouse, which is localized to two different regions of the c-myc promoter region. However, in both cases, the nuclear matrix attachment sites are close to the DNA replication origin mapped in the locus. These data suggest that global features of chromatin organization in different organisms may contribute to DNA replication origin localization.

Animals↗
PubMed1984

Initiation of SV40 DNA replication after microinjection into Xenopus eggs.

We have examined the capacity of Xenopus laevis eggs to support replication of microinjected SV40 DNA. As previously reported, microinjected DNA undergoes semi-conservative replication. Unlabeled SV40 DNA was microinjected with [3H]dTTP and, after a 3 h incubation period, the DNA was recovered and adsorbed to BND-cellulose. Elution with an NaCl gradient removes molecules that are entirely double-stranded but not those with single-stranded regions. The latter DNA population is eluted with caffeine. The radioactive DNA that eluted with NaCl was comprised mostly of supercoiled and open circular SV40 DNAs. The radioactive DNA eluted with caffeine was comprised mainly of endogenous DNA but also contained replicative forms of SV40 DNA. Analysis of SV40 DNA replication intermediates by electron microscopy revealed mainly Cairn's forms of varying degrees of maturation. Digestion with BamH1, which cleaves SV40 DNA almost opposite the normal viral replication origin, indicated that SV40 DNA microinjected into frog eggs does not initiate DNA synthesis at its normal initiation site nor at any other obvious preferred site. Rather, it appears that when this template is injected into activated Xenopus eggs, replication may initiate at random.

Animals↗
PubMedDate not supplied

Role of DNA replication in the repression of silent mating type loci in yeast.

A putative origin of DNA replication is associated with the DNA sequences necessary for the repression of silent mating type loci in yeast. These sequences lie about a kilobase away from the affected promoters, so the repression must act at a distance. We show here that DNA replication is required for the onset of repression.

Animals↗
PubMed1995

Rep protein of tomato yellow leaf curl geminivirus has an ATPase activity required for viral DNA replication.

The Rep protein of geminiviruses is the sole viral protein required for their DNA replication. The amino acid sequence of Rep protein contains an NTP binding consensus motif (P-loop). Here we show that purified Rep protein of tomato yellow leaf curl virus expressed in Escherichia coli exhibits an ATPase activity in vitro. Amino acid exchanges in the P-loop sequence of Rep causes a substantial decrease or loss of the ATPase activity. In vivo, mutant viruses carrying these Rep mutations do not replicate in plant cells. These results show that ATP binding by the Rep protein of geminiviruses is required for its function in viral DNA replication.

Adenosine Triphosphatases↗
PubMed2000

DNA replication is required To elicit cellular responses to psoralen-induced DNA interstrand cross-links.

Following introduction of DNA interstrand cross-links (ICLs), mammalian cells display chromosome breakage or cell cycle delay with a 4N DNA content. To further understand the nature of the delay, previously described as a G(2)/M arrest, we developed a protocol to generate ICLs during specific intervals of the cell cycle. Synchronous populations of G(1), S, and G(2) cells were treated with photoactivated 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and scored for normal passage into mitosis. In contrast to what was found for ionizing radiation, ICLs introduced during G(2) did not result in a G(2)/M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage and arrest in the next cell cycle. Similarly, ICLs introduced during G(1) did not cause a G(1)/S arrest. We conclude that DNA replication is required to elicit the cellular responses of cell cycle arrest and genomic instability after psoralen-induced ICLs. In primary human fibroblasts, the 4N DNA content cell cycle arrest triggered by ICLs was long lasting but reversible. Kinetic analysis suggested that these cells could remove up to approximately 2,500 ICLs/genome at an average rate of 11 ICLs/genome/h.

Bromodeoxyuridine↗