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PubMed1976

[Development of spontaneous mutations in somatic mamalian cells and DNA replication. II. Expression of gene mutations in cultured cells].

The dependence of spontaneous rate and phenotypic expression of 6-mercaptopurine resistance mutations of DNA replication synthesis was studied in cultured Chinese hamster cells. Spontaneous mutations arising with a constant rate per cell per time unit independently on DNA replication rate were shown to be expressed only in the course of subsequent cell divisions. The frequency of N-nitrosomethylurea induced mutations in cells with reduced and normal DNA replication rate is approximately the same. However, DNA replication synthesis is necessary for the phenotypic expression of both induced and spontaneous mutations. The causes of the phenotypic lag are discussed.

Animals↗
PubMed2002

S-Cdk-dependent phosphorylation of Sld2 essential for chromosomal DNA replication in budding yeast.

Cyclin-dependent protein kinases (Cdks) in eukaryotic cells work as a key enzyme at various points in the cell cycle. At the onset of S phase, active S-phase Cdks (S-Cdks) are essential for chromosomal DNA replication. Although several replication proteins are phosphorylated in a Cdk-dependent manner, the biological effects of phosphorylation of these proteins on the activation of DNA replication have not been elucidated. Here we show that Sld2 (ref. 4) (also known as Drc1; ref. 5), one of the replication proteins of budding yeast (Saccharomyces cerevisiae), is phosphorylated in S phase in an S-Cdk-dependent manner, and mutant Sld2 lacking all the preferred Cdk phosphorylation sites (All-A) is defective in chromosomal DNA replication. Moreover, the complex that contains, at least, Sld2 and Dpb11 (ref. 6) (the Sld2-Dpb11 complex) is formed predominantly in S phase; the All-A protein is defective in this complex formation. Because this complex is suggested to be essential for chromosomal DNA replication, it seems likely that S-Cdk positively regulates formation of the Sld2-Dpb11 complex and, consequently, chromosomal DNA replication.

Carrier Proteins↗
PubMed2003

Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability.

Some spontaneous gross chromosomal rearrangements (GCRs) seem to result from DNA-replication errors. The chromatin-assembly factor I (CAF-I) and replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and could play a role in maintaining genome stability. Inactivation of CAF-I or RCAF increased the rate of accumulating different types of GCRs including translocations and deletion of chromosome arms with associated de novo telomere addition. Inactivation of CAF-I seems to cause damage that activates the DNA-damage checkpoints, whereas inactivation of RCAF seems to cause damage that activates the DNA-damage and replication checkpoints. Both defects result in increased genome instability that is normally suppressed by these checkpoints, RAD52-dependent recombination, and PIF1-dependent inhibition of de novo telomere addition. Treatment of CAF-I- or RCAF-defective cells with methyl methanesulfonate increased the induction of GCRs compared with that seen for a wild-type strain. These results indicate that coupling of chromatin assembly to DNA replication and DNA repair is critical to maintaining genome stability.

Chromatin Assembly Factor-1↗
PubMed1998

p53-dependent signaling sustains DNA replication and enhances clonogenic survival in 254 nm ultraviolet-irradiated human fibroblasts.

The cyclin-dependent kinase inhibitor p21(WAF1/CIP1/SDI1/CAP20) exists in normal human fibroblasts in a quaternary complex with a cyclin, a cyclin-dependent kinase, and proliferating cell nuclear antigen. A model was proposed in which, during p53-mediated suppression of cell proliferation following treatment with 254 nm UV radiation (UVC), the enhanced expression of p21 might inhibit DNA replication by virtue of its interactions with proliferating cell nuclear antigen. To test this model, we examined the mechanisms of inhibition of DNA replication in diploid human fibroblasts that express human papillomavirus type 16 E6, which inactivates p53. E6-expressing cells were defective in G1 checkpoint responses of induction of p21 and G1 arrest after ionizing radiation-induced damage to DNA. Accordingly, E6-expressing cells were resistant to inactivation of single-cell colony formation by ionizing radiation. E6 cells also displayed normal S-phase checkpoint responses of inhibition and recovery of replicon initiation following exposure to ionizing radiation and normal ability to bypass pyrimidine dimers during DNA replication soon after UVC irradiation (i.e., postreplication repair). However, DNA replication 6 h after UVC exposure was significantly inhibited in E6 cells in comparison to isogenic controls. This failure to maintain DNA replication in S-phase cells was associated with enhanced sensitivity to inactivation of single-cell colony formation by UVC. These results indicate that the p53-induced p21 pathway is not involved in the immediate S-phase responses to radiation-induced DNA damage of inhibition of replicon initiation and translesion bypass. However, our results demonstrate that p53 and, conceivably, p21 contribute to the ability of normal human fibroblasts to sustain DNA replication activity and form colonies following UVC irradiation.

Cell Line↗
PubMed2003

The yeast Sgs1 helicase is differentially required for genomic and ribosomal DNA replication.

The members of the RecQ family of DNA helicases play conserved roles in the preservation of genome integrity. RecQ helicases are implicated in Bloom and Werner syndromes, which are associated with genomic instability and predisposition to cancers. The human BLM and WRN helicases are required for normal S phase progression. In contrast, Saccharomyces cerevisiae cells deleted for SGS1 grow with wild-type kinetics. To investigate the role of Sgs1p in DNA replication, we have monitored S phase progression in sgs1Delta cells. Unexpectedly, we find that these cells progress faster through S phase than their wild-type counterparts. Using bromodeoxyuridine incorporation and DNA combing, we show that replication forks are moving more rapidly in the absence of the Sgs1 helicase. However, completion of DNA replication is strongly retarded at the rDNA array of sgs1Delta cells, presumably because of their inability to prevent recombination at stalled forks, which are very abundant at this locus. These data suggest that Sgs1p is not required for processive DNA synthesis but prevents genomic instability by coordinating replication and recombination events during S phase.

Bromodeoxyuridine↗
PubMed1979

Relation between the SCE points and the DNA replication bands.

A method for obtaining a combination of differential sister chromatid staining and DNA replication banding is described. Using this method the SCE points can be precisely localized to particular bands of individual chromosomes. It was shown, that SCEs occur not only in the regions of early DNA replication (= euchromatic segments = negative G-bands), but also in the regions of late DNA replication bands (=heterochromatic segments=positive G-bands). SCEs occurred about three times more frequently in the euchromatic segments than in the heterochromatic segments. Furthermore, more SCEs were observed in the early replicating X-chromosome than in the late replicating X-chromosome.

Chromosome Banding↗
PubMed1987

Mode of initiation of constitutive stable DNA replication in RNase H-defective mutants of Escherichia coli K-12.

The alternative pathway of DNA replication in rnh mutants of Escherichia coli can be continuously initiated in the presence of chloramphenicol, giving rise to constitutive stable DNA replication (cSDR). We conducted a physiological analysis of cSDR in rnh-224 mutants in the presence or absence of the normal DNA replication system. The following results were obtained. cSDR allowed the cells to grow in the absence of the normal replication system at a 30 to 40% reduced growth rate and with an approximately twofold-decreased DNA content. cSDR initiation was random with respect to time in the cell cycle as well as choice of origins. cSDR initiation continued to increase exponentially for more than one doubling time when protein synthesis was inhibited by chloramphenicol. cSDR initiation was inhibited during amino acid starvation in stringent (relA+) but not in relaxed (relA1) strains, indicating its sensitivity to ppGpp. cSDR initiation was rifampin sensitive, demonstrating that RNA polymerase was involved. cSDR functioned in dnaA+ rnh-224 strains parallel to the normal oriC+ dnaA+-dependent chromosome replication system.

Amino Acids↗
PubMed1999

The effects of heat-shock on nuclear matrix-associated DNA-replication complexes.

To better understand the role of the nuclear matrix in heat-induced cell killing, we have investigated the effects of heat shock on DNA replication complexes. Changes in protein extractability are observed following heat shock, including stabilization of which stabilize DNA replication complexes in association with the nuclear matrix. This situation is accompanied by differential delays in the progress and completion of DNA synthesis and the transition from type I to type II DNA replication patterns. Interestingly, prolonged delays in restarting DNA synthesis produced significant protection from heat-induced cell killing. These results show that nuclear matrix-associated DNA replication complexes may be important targets for heat-induced cell killing.

Animals↗
PubMed2005

Chromatin remodeling factors and DNA replication.

Chromatin structures have to be precisely duplicated during DNA replication to maintain tissue-specific gene expression patterns and specialized domains, such as the centromeres. Chromatin remodeling factors are key components involved in this process and include histone chaperones, histone modifying enzymes and ATP-dependent chromatin remodeling complexes. Several of these factors interact directly with components of the replication machinery. Histone variants are also important to mark specific chromatin domains. Because chromatin remodeling factors render chromatin dynamic, they may also be involved in facilitating the DNA replication process through condensed chromatin domains.

Adenosine Triphosphatases↗
PubMed1975

Biogenesis of mitochondria. 43. A comparative study of petite induction and inhibition of mitochondrial DNA replication in yeast by ethidium bromide and berenil.

The action of ethidium bromide and berenil on the mitochondrial genome of Saccharomyces cerevisiae has been compared in three types of study: (i) early kinetics (up to 4 h) of petite induction by the drugs in the presence or absence of sodium dodecyl sulphate; (ii) genetic consequences of long-term (8 cell generations) exposure to the drugs; (iii) inhibition of mitochondrial DNA replication, both in whole cells and in isolated mitochondria. The results have been interpreted as follows. Firstly, the early events in petite induction differ markedly for the two drugs, as indicated by differences in the short-term kinetics. After some stage a common pathway is apparently followed because the composition of the population of petite cells induced after long-term exposure are very similar for both ethidium bromide and berenil. Secondly, both drugs probably act at the same site to inhibit mitochondrial DNA replication, in view of the fact that a petite strain known to be resistant to ethidium bromide inhibition of mitochondrial DNA replication was found to have simultaneously acquired resistance to berenil. From consideration of the drug concentrations needed to inhibit mitochondrial DNA replication in vivo and in vitro it is suggested that in vivo permeability barriers impede the access of ethidium bromide to the site of inhibition of mitochondrial DNA replication, whilst access of berenil to this site is facilitated. The site at which the drugs act to inhibit mitochondrial DNA replication may be different from the site(s) involved in early petite induction. Binding of the drugs at the latter site(s) is considered to initiate a series of events leading to the fragmentation of yeast mitochondrial DNA and petite induction.

Amidines↗
PubMed1994

The stimulatory effect of nickel chloride on DNA replication in human HeLa cells and Escherichia coli.

An investigation was undertaken to study DNA replication in cultured human HeLa cells and Escherichia coli in response to nickel chloride (NiCl2). Treatment with NiCl2 increased both the rate of DNA replication and total cell number in HeLa cells and E. coli in a time- and concentration-dependent manner. The maximum stimulation of thymidine uptake into DNA was observed with 0.125-0.25 mM NiCl2 for both cell types. In studies of DNA replication using a crude HeLa cellular extract, NiCl2 at concentrations below 0.125 mM also induced a stimulation over the background of MgCl2-dependent [3H]dTMP incorporation into activated calf thymus DNA. However, a similar stimulatory effect from NiCl2 was not observed with either purified HeLa DNA polymerase alpha or E.coli DNA polymerase I Klenow fragment. In the absence of Mg2+, the low response of either DNA polymerase alpha or Klenow fragment to stimulation by Ni2+ was thought to be enhanced by the presence of Ni(2+)-binding proteins presented in the crude HeLa cell extract.

Cell Division↗
PubMed2005

TATA-binding protein and TBP-associated factors during herpes simplex virus type 1 infection: localization at viral DNA replication sites.

Host RNA polymerase II (RNAP II) is responsible for viral transcription of the herpes simplex virus type 1 (HSV-1) genome and is relocalized to viral DNA replication compartments. Thus, we investigated whether TATA-binding protein (TBP) and TBP-associated factors (TAFs) are recruited to sites of viral transcription and replication and whether TBP/TAF expressions are influenced upon infection. The protein levels of TBP, hsTAF1/TAF(II)250, hsTAF4/TAF(II)135, and hsTAF5/TAF(II)100 were constant during the early phase of infection and started to decrease late during infection. Only for hsTAF7/TAF(II)55 we sometimes observed a decrease already at 4-8h postinfection (p.i.). Concomitantly with the relocalization of RNAP II, TBP and hsTAFs were redistributed to sites of viral DNA replication and transcription. In the absence of viral DNA replication TBP/hsTAFs were present in distinct nuclear dots, however, enlargement of the nuclear structures did not take place. Our results show that HSV-1 infection has no influence on the protein levels of TFIID components and leads to a redistribution of TBP and hsTAFs to prereplicative sites that enlarge to viral DNA replication compartments.

Cell Nucleus↗
PubMed2002

Biased incorporation of ribonucleotides on the mitochondrial L-strand accounts for apparent strand-asymmetric DNA replication.

Recently, we presented evidence for conventional, strand-coupled replication of mammalian mitochondrial DNA. Partially single-stranded replication intermediates detected in the same DNA preparations were assumed to derive from the previously described, strand-asymmetric mode of mitochondrial DNA replication. Here, we show that bona fide replication intermediates from highly purified mitochondria are essentially duplex throughout their length, but contain widespread regions of RNA:DNA hybrid, as a result of the incorporation of ribonucleotides on the light strand which are subsequently converted to DNA. Ribonucleotide-rich regions can be degraded to generate partially single-stranded molecules by RNase H treatment in vitro or during DNA extraction from crude mitochondria. Mammalian mitochondrial DNA replication thus proceeds mainly, or exclusively, by a strand-coupled mechanism.

Animals↗
PubMed1998

Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells.

Cdc6 has a critical regulatory role in the initiation of DNA replication in yeasts, but its function in mammalian cells has not been characterized. We show here that Cdc6 is expressed selectively in proliferating but not quiescent mammalian cells, both in culture and within tissues of intact animals. During the transition from a growth-arrested to a proliferative state, transcription of mammalian Cdc6 is regulated by E2F proteins, as revealed by a functional analysis of the human Cdc6 promoter and by the ability of exogenously expressed E2F proteins to stimulate the endogenous Cdc6 gene. Immunodepletion of Cdc6 by microinjection of anti-Cdc6 antibody blocks initiation of DNA replication in a human tumor cell line. We conclude that expression of human Cdc6 is regulated in response to mitogenic signals though transcriptional control mechanisms involving E2F proteins, and that Cdc6 is required for initiation of DNA replication in mammalian cells.

Animals↗
PubMed1999

In vitro DNA replication in yeast nuclear extracts.

Two assays have been developed for studying DNA replication in vitro based on nuclear extracts isolated from budding yeast cells synchronized in S phase. In the first, the template DNA for replication is provided in the form of intact yeast nuclei, usually from cells arrested in G(1). In the second assay, bacterially produced supercoiled plasmid is replicated in an S-phase nuclear extract supplemented with nucleotides and an energy-regenerating system. Semiconservative DNA replication is monitored by substitution of newly synthesized DNA with bromodeoxyuridine 5'-triphosphate (BrdUTP) and density gradient analysis. In addition, neutral-neutral two-dimensional gel analyses and, in the case of nuclei, detection of newly synthesized DNA in replication foci by DIG-dUTP incorporation can be used to monitor replication.

Cell Nucleus↗
PubMed1999

9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp) as a substrate toward replicative DNA polymerases alpha, delta, epsilon, and epsilon*.

The diphosphoryl derivative of the acyclic nucleotide phosphonate analog 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), found previously to weakly inhibit DNA pol delta/proliferating cell nuclear antigen, was studied as a substrate for pol alpha, delta, epsilon, and epsilon*. A comparison of the Vmax and Km for this derivative (PMEApp) and dATP demonstrated that the relative efficiency of the incorporation of this analog into the DNA chain is decreasing in the following order: pol delta approximately equal to pol epsilon approximately equal to pol epsilon* > pol alpha. Under the reaction conditions, this incorporation amounted to 4.4 to 0.7% of dAMP molecules. Similar Km values for PMEApp and dATP in pol epsilon and pol epsilon* catalyzed reactions revealed that proteolysis of the enzyme probably does not affect the dNTP binding site. The DNA polymerases tested were inhibited by the reaction product (PMEA terminated DNA chain) with similar Ki/Km ratios (pol alpha 0.2; pol delta, 0.1; pol epsilon 0.05; and pol epsilon*, 0.06). The associated 3'-5'-exonuclease activity of pol delta, epsilon, and epsilon* was able to excise PMEA from the 3'-OH end of DNA with a rate one order of magnitude lower than that of the dAMP residue.

Adenine↗