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PubMed2002

Relationship between vaccinia virus intracellular cores, early mRNAs, and DNA replication sites.

Virus assembly, a late event in the life cycle of vaccinia virus (VV), is preceded by a number of steps that all occur in the cytoplasm of the infected host cell: virion entry, delivery of the viral core into the cytoplasm, and transcription from these cores of early mRNAs, followed by the process of DNA replication. In the present study the quantitative and structural relationships between these distinct steps of VV morphogenesis were investigated. We show that viral RNA and DNA synthesis increases linearly with increasing amounts of incoming cores. Moreover, at multiplicities of infection that result in 10 to 40 cores per cell, an approximately 1:1 ratio between cores and sites of DNA replication exists, suggesting that each core is infectious. We have shown previously that VV early mRNAs collect in distinct granular structures that recruit components of the host cell translation machinery. Strikingly, these structures appeared to form some distance away from intracellular cores (M. Mallardo, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:3875-3891, 2001). In the present study the intracellular locations of the sites of early mRNA accumulation and those of the subsequent process of DNA replication were compared. We show that these are distinct structures that have different intracellular locations. Finally, we study the fate of the parental DNA after core uncoating. By electron microscopy, cores were found close to membranes of the endoplasmic reticulum (ER) and the parental DNA, once it had left the core, appeared to associate preferentially with the cytosolic side of those membranes. Since we have previously shown that the process of DNA replication occurs in an ER-enclosed cytosolic "subcompartment" (N. Tolonen, L. Doglio, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:2031-2046, 2001), the present data suggest that the parental DNA is released into the cytosol and associates with the same membranes where DNA replication is subsequently initiated. The combined data are discussed with respect to the cytosolic organization of VV morphogenesis.

Cytoplasm↗

PubMed1997

Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants.

Faithful inheritance of genetic information requires that DNA be copied only once each cell cycle. Initiation of DNA replication involves the establishment of a prereplication complex (pre-RC) and subsequent activation by CDK/cyclins, converting the pre-RC to a post-RC. The origin recognition complex (ORC), Cdc6p, and the MCM proteins are required for establishing the pre-RC. We show that all six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. A newly isolated cdc6 mutant displays promiscuous initiation of DNA replication, increased nuclear DNA content, and constant MCM protein association with chromatin throughout the cell cycle. This gain-of-function cdc6 mutant ignores the negative controls imposed normally on initiation by the CDK/cyclins, suggesting that Cdc6p is a key mediator of once-per-cell-cycle control of DNA replication.

CDC28 Protein Kinase, S cerevisiae↗

PubMed2005

Chk1 and p21 cooperate to prevent apoptosis during DNA replication fork stress.

Cells respond to DNA replication stress by triggering cell cycle checkpoints, repair, or death. To understand the role of the DNA damage response pathways in determining whether cells survive replication stress or become committed to death, we examined the effect of loss of these pathways on cellular response to agents that slow or arrest DNA synthesis. We show that replication inhibitors such as excess thymidine, hydroxyurea, and camptothecin are normally poor inducers of apoptosis. However, these agents become potent inducers of death in S-phase cells upon small interfering RNA-mediated depletion of the checkpoint kinase Chk1. This death response is independent of p53 and Chk2. p21-deficient cells, on the other hand, produce a more robust apoptotic response upon Chk1 depletion. p21 is normally induced only late after thymidine treatment. In Chk1-depleted cells p21 induction occurs earlier and does not require p53. Thus, Chk1 plays a primary role in the protection of cells from death induced by replication fork stress, whereas p21 mediates through its role in regulating entry into S phase. These findings are of potential importance to cancer therapy because we demonstrate that the efficacy of clinically relevant agents can be enhanced by manipulation of these signaling pathways.

Apoptosis↗

PubMed2001

DNA replication control through interaction of E2F-RB and the origin recognition complex.

The E2F transcription factor and retinoblastoma protein control cell-cycle progression and DNA replication during S phase. Mutations in the Drosophila dE2F1 and dDP genes affect the origin recognition complex (DmORC) and initiation of replication at the chorion gene replication origin. Here we show that mutants of Rbf (an retinoblastoma protein homologue) fail to limit DNA replication. We also show that the dDP, dE2F1 and Rbf proteins are located in a complex with DmORC, and that dE2F1 and DmORC are bound to the chorion origin of replication in vivo. Our results indicate that dE2F1 and Rbf function together at replication origins to limit DNA replication through interactions with DmORC.

Animals↗

PubMed1996

Characterization of an essential Orc2p-associated factor that plays a role in DNA replication.

The Saccharomyces cerevisiae Orc2 protein is a subunit of the origin recognition complex, ORC, which binds in a sequence-specific manner to yeast origins of DNA replication. With screens for orc2-1 synthetic lethal mutations and Orc2p two-hybrid interactors, a novel Orc2p-associated factor (Oaf1p) was identified. OAF1 is essential, its gene product is localized to the nucleus, and an oaf1 temperature-sensitive mutant arrests as large budded cells with a single nucleus. The mutant oaf1-2, isolated in the synthetic lethal screen, loses plasmids containing a single origin of DNA replication at a high rate, but it maintains plasmids carrying multiple potential origins of DNA replication. In addition, the OAF1 gene product tagged with the hemagglutinin antigen epitope binds to a DNA affinity column containing covalently linked tandem repeats of an essential origin element. These results suggest a role for OAFI in the initiation of DNA replication. Mutant alleles of cdc7 and cdc14 were also isolated in the orc2-1 synthetic lethal screen. Cdc7p, like Oaf1p, also interacts with Orc2p in two-hybrid assays.

Amino Acid Sequence↗

PubMed2006

Functional requirement of noncoding Y RNAs for human chromosomal DNA replication.

Noncoding RNAs are recognized increasingly as important regulators of fundamental biological processes, such as gene expression and development, in eukaryotes. We report here the identification and functional characterization of the small noncoding human Y RNAs (hY RNAs) as novel factors for chromosomal DNA replication in a human cell-free system. In addition to protein fractions, hY RNAs are essential for the establishment of active chromosomal DNA replication forks in template nuclei isolated from late-G(1)-phase human cells. Specific degradation of hY RNAs leads to the inhibition of semiconservative DNA replication in late-G(1)-phase template nuclei. This inhibition is negated by resupplementation of hY RNAs. All four hY RNAs (hY1, hY3, hY4, and hY5) can functionally substitute for each other in this system. Mutagenesis of hY1 RNA showed that the binding site for Ro60 protein, which is required for Ro RNP assembly, is not essential for DNA replication. Degradation of hY1 RNA in asynchronously proliferating HeLa cells by RNA interference reduced the percentages of cells incorporating bromodeoxyuridine in vivo. These experiments implicate a functional role for hY RNAs in human chromosomal DNA replication.

Animals↗

PubMed1987

Bacteriophage P4 DNA replication. Nucleotide sequence of the P4 replication gene and the cis replication region.

A 3100 base piece of DNA from the 11,500 base genome of bacteriophage P4 was analyzed for its nucleotide sequence. This segment of DNA contains two open reading frames of 106 and 777 amino acid residues; the latter of which is the coding sequence for the Mr 84,841 alpha protein, which is necessary for P4 DNA replication and is thought to act as a P4-specific DNA primase. A region of about 300 base-pairs localized just beyond the alpha gene and about 4500 bases from the origin of replication (ori), was defined as the locus for P4's cis replication region (crr). This region is required for replication both in vivo and in vitro, and consists of two directly repeated sequences of 120 base-pairs that match one another at 98 positions. These directly repeated sequences are separated by 60 base-pairs, which are not necessary for replication. Each repeat in crr contains three copies of the octamer TGTTCACC that is found six times in ori. Either of the 120 base-pair repeat sequences in crr is sufficient for replication, and the entire crr can function in an inverted orientation. crr is also active at a distance of 1800 bases from the P4 origin of replication.

Bacteriophages↗

PubMed1990

Localization of adenovirus-encoded DNA replication proteins in the nucleus by immunogold electron microscopy.

The distribution of three adenovirus-encoded DNA replication proteins in the nucleus of human 293 cells was studied by immunogold electron microscopy. The infected nuclei contained four morphologically distinct inclusions. They were highly electron-dense granules (type I), compact fibrogranular masses of medium electron density (type II), filamentous masses of low electron density (type III) and large polygonal crystals (type IV). In immunogold labelling studies, antibodies to the adenovirus single-stranded DNA-binding protein (DBP) and antibodies to single-stranded DNA showed extensive binding to the type III inclusions. The antibodies to the adenovirus DNA polymerase (AdPol) and terminal protein (TP) predominantly labelled type II inclusions. Double immunogold labelling studies detected low levels of AdPol and TP in type III inclusions and DBP in type II inclusions. The selective distribution of DNA replication proteins suggests that the type II and III inclusions represent two functionally different entities that may be involved in two different aspects of adenovirus DNA replication, i.e. chain initiation and elongation.

Adenoviridae↗

PubMed1988

Derepression of a novel class of vaccinia virus genes upon DNA replication.

A novel class of vaccinia virus genes, called intermediate, is expressed immediately post-replication and prior to the onset of late gene transcription. Intermediate transcription is dependent on trans-acting factors which are present in an active state in virus-infected cells prior to the onset of DNA replication. Plasmid-borne intermediate genes transfected into vaccinia-virus infected cells are expressed prior to DNA replication, whereas the copies within the viral genome are repressed. DNA replication is essential for activation of viral intermediate transcription and de novo protein synthesis is not required post-replication. In contrast, activation of late transcription depends on DNA replication and continued de novo protein synthesis. Therefore, a subset of intermediate proteins is likely to be trans-activators of late gene transcription. Cell-free extracts differentially transcribe early, intermediate and late genes in a way similar to the temporal expression observed in vivo. A cascade model is discussed for the regulation of gene expression during the viral life-cycle.

Base Sequence↗

PubMed1997

Histone H1 modulates DNA replication through multiple pathways in Xenopus egg extract.

We investigated the effects of histone H1s on DNA replication using Xenopus egg extract. Mouse variants H1c and H10 were assembled onto Xenopus sperm chromatin by the extract during the remodeling that accompanies nuclear decondensation. The association of H1 with chromatin was rapid and concentration dependent. H1-associated chromatin displayed a typical nucleosomal repeat pattern indicating that linker histones are properly positioned along the DNA. The presence of H1 on sperm chromatin reduced both the rate and extent of DNA replication in egg extract. This reduction in rate is due, in part, to a delay in initiation of replication within individual nuclei. Initiation in extract is dependent upon nuclear assembly. Analysis of the assembly process revealed that H1 does not inhibit nuclear membrane formation or the import of nuclear protein, however, it does slow the rate of nuclear lamina formation. This H1-induced delay in lamina assembly is responsible for the delay in initiation as pre-assembled H1-containing nuclei initiate replication at the same time as control nuclei. However, H1 inhibits replication even when lamina assembly is complete suggesting that H1 also affects replication directly. These data indicate that H1 modulates DNA replication through multiple pathways in egg extract.

Animals↗

PubMed1994

Purification and characterization of bacteriophage T4 gene 59 protein. A DNA helicase assembly protein involved in DNA replication.

The T4 bacteriophage gene 59 protein is required for normal T4 DNA replication. We have purified this protein to homogeneity in two steps and show that it binds both to single-stranded DNA and to the T4 gene 32 protein, a DNA single strand binding protein. In in vitro assays, covering DNA with 32 protein makes this DNA inaccessible to the 41 protein, the highly processive DNA helicase, that associates with the T4 DNA primase (gene 61 protein) to form an active primosome. However, the 59 protein brings about the rapid assembly of 41 protein onto single-stranded DNA, even if this DNA is covered with 32 protein. The 59 protein is therefore a DNA helicase assembly protein. The observed requirements for the 59 protein in the vivo T4 DNA replication are explained by there being two alternative pathways for loading the 41 protein onto a replication fork at early times of T4 DNA synthesis, with only a 59 protein-mediated pathway remaining operative for the recombination-mediated replication that dominates later in infection (Barry, J., and Alberts, B. M. (1994) J. Biol. Chem. 269, 33063-33068).

Adenosine Triphosphate↗

PubMed1979

Effect of novobiocin on initiation of DNA replication in Bacillus subtilis.

The initiation of DNA replication of small replicons in vitro involves conformational changes in the whole DNA molecule or in the region near to the replication origin. One striking finding has been the role of DNA gyrase (that is, the necessity for supercoiled structure) in the initial stage of ColE1 replication in vitro. However, little is known about the effect of gyrase on the initiation of replication of bacterial chromosomes in vivo. We have constructed a map of cleavage sites of restriction enzymes at the region of the origin of replication of the Bacillus subtilis chromosome (accompanying paper). This has now enabled us to examine the effect of novobiocin, a selective inhibitor of DNA gyrase, on the replication of the specific chromosomal segments near the origin and to seek a possible role for the gyrase in the initiation of chromosomal replication. We have found that only a limited segment of the chromosome at the origin region was replicated in the presence of novobiocin. This effect allowed us to locate the site of the origin of replication to within a DNA fragment of molecular weight 3.4 x 10(6).

Bacillus subtilis↗

PubMed2001

The Saccharomyces cerevisiae MUM2 gene interacts with the DNA replication machinery and is required for meiotic levels of double strand breaks.

The Saccharomyces cerevisiae MUM2 gene is essential for meiotic, but not mitotic, DNA replication and thus sporulation. Genetic interactions between MUM2 and a component of the origin recognition complex and polymerase alpha-primase suggest that MUM2 influences the function of the DNA replication machinery. Early meiotic gene expression is induced to a much greater extent in mum2 cells than in meiotic cells treated with the DNA synthesis inhibitor hydroxyurea. This result indicates that the mum2 meiotic arrest is downstream of the arrest induced by hydroxyurea and suggests that DNA synthesis is initiated in the mutant. Genetic analyses indicate that the recombination that occurs in mum2 mutants is dependent on the normal recombination machinery and on synaptonemal complex components and therefore is not a consequence of lesions created by incompletely replicated DNA. Both meiotic ectopic and allelic recombination are similarly reduced in the mum2 mutant, and the levels are consistent with the levels of meiosis-specific DSBs that are generated. Cytological analyses of mum2 mutants show that chromosome pairing and synapsis occur, although at reduced levels compared to wild type. Given the near-wild-type levels of meiotic gene expression, pairing, and synapsis, we suggest that the reduction in DNA replication is directly responsible for the reduced level of DSBs and meiotic recombination.

Alleles↗

PubMed2004

RB reversibly inhibits DNA replication via two temporally distinct mechanisms.

The retinoblastoma (RB) tumor suppressor is a critical negative regulator of cellular proliferation. Repression of E2F-dependent transcription has been implicated as the mechanism through which RB inhibits cell cycle progression. However, recent data have suggested that the direct interaction of RB with replication factors or sites of DNA synthesis may contribute to its ability to inhibit S phase. Here we show that RB does not exert a cis-acting effect on DNA replication. Furthermore, the localization of RB was distinct from replication foci in proliferating cells. While RB activation strongly attenuated the RNA levels of multiple replication factors, their protein expression was not diminished coincident with cell cycle arrest. During the first 24 h of RB activation, components of the prereplication complex, initiation factors, and the clamp loader complex (replication factor C) remained tethered to chromatin. In contrast, the association of PCNA and downstream components of the processive replication machinery was specifically disrupted. This signaling from RB occurred in a manner dependent on E2F-mediated transcriptional repression. Following long-term activation of RB, we observed the attenuation of multiple replication factors, the complete cessation of DNA synthesis, and impaired replicative capacity in vitro. Therefore, functional distinctions exist between the "chronic" RB-mediated arrest state and the "acute" arrest state. Strikingly, attenuation of RB activity reversed both acute and chronic replication blocks. Thus, continued RB action is required for the maintenance of two kinetically and functionally distinct modes of replication inhibition.

Alleles↗

PubMed1983

Mode of inhibition of DNA replication in neocarzinostatin-treated HeLa cells.

The effect of antitumor antibiotic neocarzinostatin on DNA replication in HeLa cells was studied by pulse-labeling of DNA with [3H]thymidine and sedimentation analysis of the DNA with alkaline sucrose gradients. The drug, which produced DNA damage, primarily inhibited the replicon initiation in the cells at low doses (less than or equal to 0.1 microgram/ml), and at high doses (greater than or equal to 0.5 microgram/ml) inhibited the DNA chain elongation. An analysis of the number of single-strand breaks of parental DNA, induced by neocarzinostatin, indicated that inhibition of the initiation occurred with introduction of single-strand breaks of less than 1.5 . 10(4)/cell, while inhibition of the elongation occurred with introduction of single-strand breaks of more than 7.5 . 10(4)/cell. Assuming that the relative molecular mass of DNA/HeLa cell was about 10(13) Da, the target size of DNA for inhibition of replicon initiation was calculated to be about 10(9) Da, such being close to an average size of loop DNA in the cell and for inhibition of chain elongation, 1-2 . 10(8) Da which was of the same order of magnitude as the size of replicons. Recovery of inhibited DNA replication by neocarzinostatin occurred during post-incubation of the cells and seemed to correlate with the degree of rejoining of the single-strand breaks of parental DNA. Caffeine and theophylline enhanced the recovery of the inhibited replicon initiation, but did not aid in the repair of the breaks in parental DNA.

Antibiotics, Antineoplastic↗

PubMed2007

No single homologous repeat region is essential for DNA replication of the baculovirus Autographa californica multiple nucleopolyhedrovirus.

The presence of homologous repeat (hr) regions in multiple locations within baculovirus genomes has led to the hypothesis that they represent origins of DNA replication. This hypothesis has been supported by transient replication assays where plasmids carrying hrs replicated in the presence of virus DNA replication. This study investigated whether any specific hr region was essential for viral DNA replication in vivo, by generating a series of recombinant Autographa californica multiple nucleopolyhedrovirus where the lacZ gene replaced hr1, hr1a, hr2, hr3, hr4a or hr4b. In addition, a double-hr knockout virus was constructed where both hr2 and hr3 were deleted. The successful construction of these knockout viruses indicated that no specific region was essential for virus production. These recombinant viruses were characterized by titrations of budded virus, expression of a variety of virus-specific proteins and the synthesis of viral DNA at various times after infection. The results demonstrated that each hr was dispensable for all of these properties and that no single region was absolutely essential for virus replication in cell culture. The functional significance of multiple origin regions is still unclear.

Animals↗

PubMed1997

Requirements for Epstein-Barr nuclear antigen 1 (EBNA1)-induced permanganate sensitivity of the epstein-barr virus latent origin of DNA replication.

Epstein-Barr nuclear antigen 1 (EBNA1) activates DNA replication from the Epstein-Barr virus latent origin of DNA replication, oriP. EBNA1 binds cooperatively to four recognition sites in the dyad symmetry (DS) element of oriP, causing alterations in the origin DNA structure, which can be detected by the increased sensitivity of one Thy residue in two of the binding sites to permanganate oxidation. To better understand the significance of this EBNA1-induced origin distortion, we have investigated the DNA sequence and EBNA1 amino acid requirements for permanganate sensitivity. We have shown that the EBNA1 DNA binding and dimerization domains are sufficient to induce permanganate sensitivity and that amino acids 463-467, which form an extended chain that travels along the minor groove of the EBNA1 recognition site, play an important role in generating the DNA distortion. The EBNA1-induced permanganate sensitivity is independent of cooperative interactions between EBNA1 molecules on the origin and requires a specific sequence within the EBNA1 binding site. Using synthetic EBNA1 binding sites, we found that the inversion of a single AT base pair in the EBNA1 recognition sequence is sufficient to confer EBNA1-induced permanganate sensitivity. These studies indicate that permanganate oxidation can detect very minor alterations in DNA structure.

Binding Sites↗

PubMed1981

DNA replication of X-irradiated human lymphocytes.

Human peripheral blood lymphocytes are well-differentiated cells. Ordinarily, they do not divide and are considered to be in the G0 stage of the cell cycle. These cells can be stimulated to undergo DNA replication in culture by mitogens such as phytohemagglutinin. In the present study, we have examined cellular and biochemical events that occur after exposure of lymphocytes to X-irradiation. Irradiation with up to 100 rads, prior to stimulation with phytohemagglutinin, did not interfere with DNA replication. At later periods, DNA replication was inhibited proportionally to the amount of radiation. In comparison to DNA synthesis, the effect of X-irradiation on RNA and protein synthesis in phytohemagglutinin-stimulated lymphocytes was less marked. Furthermore, X-rays did not inhibit either the induction or the continual synthesis of DNA polymerase-alpha or -beta in response to phytohemagglutinin. Kinetic studies with different nucleotide substrates suggest that cellular pools of nucleotides are not significantly altered by X-irradiation. Thus, the inhibition of DNA synthesis in irradiated cells is likely to be due to damage to the cellular DNA template. The inhibition of DNA synthesis was accompanied by accumulation of cells in the G2 and M stages of the cell cycle, suggesting that inhibition of DNA replication by X-irradiation is a postmitotic event.

Cell Cycle↗