Relationship of single-stranded DNA-binding proteins of Ehrlich ascites tumour to cell growth phase and DNA replications.

The possible involvement of SSB-proteins in DNA replication in Ehrlich ascites tumour (EAT) has been investigated. A direct relation (the computer-generated correlation coefficient was 0.9) between the SSB-proteins content in chromatin and intensity of the replicative synthesis of DNA in various preparation of EAT in vivo and in vitro is observed. Addition of exogenous SSB-proteins to the permeable EAT cells has been found to increase the replicative synthesis. Although eukaryotic SSB-proteins are not complete analogs of the prokaryotic SSB-proteins, they evidently participate in DNA replication in eukaryotic cells and possibly are intracellular regulators of proliferation.     

Stimulation of replicative DNA synthesis by eukaryotic proteins bound to single-stranded DNA]

The paper deals with the effect of the single-strand (ss) DNA-binding proteins (SSB-proteins) from the Ehrlich ascites tumor (EAT) cells and from the eggs of silkworm, as well as the mouse serum blood proteins, having preferential affinity to ss DNA, on the DNA replicative synthesis in the EAT cells permeable for the macromolecules, and, for the silkworm proteins and on the DNA replicative synthesis in the nuclei from the eggs of silkworm proteins and on the DNA replicative synthesis in the nuclei from the eggs of silkworm permeable for macromolecules. SSB-proteins of EAT to considerable extent stimulated the DNA synthesis. At the same time, the other proteins (from the silkworm and from the serum) activated the DNA synthesis in the permeable cells to the less extent. It was found that SSB-proteins from the silkworm had a 1.5-13 fold stimulating effect on the DNA replicative synthesis in the homologous system (in the permeable nuclei). If the permeability for the macromolecules of the cells and nuclei treatment with Triton X-100 may be different, it is supposed that the activation of the DNA synthesis by the exogenous proteins depends on the homologous system of the DNA replicative complex. It is possible that the effect of the serum proteins on the DNA synthesis is connected with the masking of the ss regions of DNA which inhibited DNA-polymerase alpha. Perhaps the mechanisms of the activation of the DNA replicative synthesis by the proteins in vitro with the purified DNA polymerase alpha and in vivo are of different nature and are conditioned by homology of the deoxyribonucleoproteins.     

Use of benzoyl-naphthyl-DEAE-cellulose chromatography for determining the ability of proteins from Ehrlich ascites carcinoma, binding with single-stranded DNA (SSB-proteins), to destabilize the DNA double helix]

Single-stranded DNA-binding proteins (SSB-proteins) isolated from Ehrlich ascites tumour (EAT) cells were incubated for 30 min at 5 mM NaCl with salmon sperm DNA or [3H]DNA from EAT at the SSB-protein/DNA ratio (w/w) of 0 to 4.5. After addition of sodium dodecyl sulfate up to a 0.05% concentration, the proteins were applied to columns with benzoylated naphthoylated DEAE-cellulose. Double-stranded DNA was eluted by 1 M NaCl; the DNA containing single-stranded regions was eluted by 50% dimethylformamide. There was a progressive lowering of the DNA content in the first eluate and a rise in the second eluate, as could be evidenced from the increase in the SSB-protein/DNA w/w ratio. This effect was more pronounced in the case of homologous DNA and was not coupled with the nuclease activity of SSB proteins. It was concluded that EAT SSB-proteins are "DNA-unwinding" proteins.     

The effect of SSB-protein from Ehrlich ascites carcinoma cells on activity of various enzymes of DNA replication and repair

The effect of a single-stranded DNA-binding protein (SSB-protein) form Ehrlich ascites tumour cells (EAT) on the activity of homologous purified DNA-polymerases alpha and beta, DNA-replicase, primase and DNA-polymerases from phage T4 and Bacillus stearothermophillus was studied. It was shown that the SSB-protein caused a 1.5-2.5-fold stimulation of the DNA-polymerase alpha activity on different templates (e.g., denaturated and activated DNA, poly(dA). The degree of stimulation depended on the template type, protein/template ratio and purity of DNA-polymerase alpha. The activity of DNA-polymerase was inhibited by the SSB-protein, when the activated DNA was used as a matrix and was unchanged on the denaturated DNA. The activity of some prokaryotic DNA-polymerases was increased under the influence of the SSB-protein. The protein enhanced the processivity of T4 DNA-polymerase and strongly inhibited the activity of replicase and primase. A conclusion about the complex effect of the SSB-protein on the activity of replicative and repair enzymes is drawn.     

A gene that regulates DNA replication in response to DNA damage is located on human chromosome 4q.

Inhibition of replicative DNA synthesis following gamma-irradiation is observed in eukaryotic cells but is defective in cells derived from patients with the cancer-prone inherited disorder ataxia-telangiectasia (A-T) and in A-T-like Chinese hamster cell mutants. Chinese hamster cells show a less pronounced inhibition of DNA synthesis after gamma-irradiation when compared to irradiated human HeLa or mouse A9 cells. Therefore, to identify new human genes involved in the regulation of DNA replication in response to ionizing radiation in mammalian cells, single human chromosomes were introduced into Chinese hamster cells by microcell-mediated chromosome transfer. It is found that a new gene on human chromosome 4q inhibits DNA synthesis following gamma- and UV irradiation in hamster cells. However, this delay of DNA replication did not improve cell survival or the level of chromosomal aberrations induced by X-rays, indicating that the lack of the inhibition of DNA synthesis after X-irradiation is not a prerequisite for the X-ray sensitivity and chromosomal instability, which is observed in A-T and A-T-like hamster cells.     

Interaction of human DNA polymerase eta with monoubiquitinated PCNA: a possible mechanism for the polymerase switch in response to DNA damage

Most types of DNA damage block replication fork progression during DNA synthesis because replicative DNA polymerases are unable to accommodate altered DNA bases in their active sites. To overcome this block, eukaryotic cells employ specialized translesion synthesis (TLS) polymerases, which can insert nucleotides opposite damaged bases. In particular, TLS by DNA polymerase eta (poleta) is the major pathway for bypassing UV photoproducts. How the cell switches from replicative to TLS polymerase at the site of blocked forks is unknown. We show that, in human cells, PCNA becomes monoubiquitinated following UV irradiation of the cells and that this is dependent on the hRad18 protein. Monoubiquitinated PCNA but not unmodified PCNA specifically interacts with poleta, and we have identified two motifs in poleta that are involved in this interaction. Our findings provide an attractive mechanism by which monoubiquitination of PCNA might mediate the polymerase switch.     

Involvement of DnaE, the second replicative DNA polymerase from Bacillus subtilis, in DNA mutagenesis

In a large group of organisms including low G + C bacteria and eukaryotic cells, DNA synthesis at the replication fork strictly requires two distinct replicative DNA polymerases. These are designated pol C and DnaE in Bacillus subtilis. We recently proposed that DnaE might be preferentially involved in lagging strand synthesis, whereas pol C would mainly carry out leading strand synthesis. The biochemical analysis of DnaE reported here is consistent with its postulated function, as it is a highly potent enzyme, replicating as fast as 240 nucleotides/s, and stalling for more than 30 s when encountering annealed 5'-DNA end. DnaE is devoid of 3' --> 5'-proofreading exonuclease activity and has a low processivity (1-75 nucleotides), suggesting that it requires additional factors to fulfill its role in replication. Interestingly, we found that (i) DnaE is SOS-inducible; (ii) variation in DnaE or pol C concentration has no effect on spontaneous mutagenesis; (iii) depletion of pol C or DnaE prevents UV-induced mutagenesis; and (iv) purified DnaE has a rather relaxed active site as it can bypass lesions that generally block other replicative polymerases. These results suggest that DnaE and possibly pol C have a function in DNA repair/mutagenesis, in addition to their role in DNA replication.     

Participation of the SSB protein in the repair of the DNA of Ehrlich ascites carcinoma cells following UV irradiation

Synchronous changes were detected in the SSB-protein content of the chromatin and in the rate of repair DNA synthesis at different time intervals after UV-irradiation of Ehrlich ascites tumor cells. The amount of SSB-protein in the extra-chromatin fraction was in an inverse relation to its content in the chromatin, whereas the cumulative SSB-protein content remained invariable. Similar changes in the SSB-protein content of the chromatin and in repair synthesis were also registered after the effect of various doses of UV-light. The increase of the SSB-protein content in the chromatin was not connected with the postirradiation accumulation of single-strand sites in DNA.     

A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication

In vertebrates, MCM2-7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2-7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2-7 unwinds DNA during replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication. A fragment of the retinoblastoma protein, Rb(1-400), was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb(1-400) and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2-7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor.     

Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA

Proliferating cell nuclear antigen (PCNA) is a cell cycle and growth regulated protein required for replication of SV40 DNA in vitro. Its function was investigated by comparison of the replication products synthesized in its presence or absence. In the completely reconstituted replication system that contains PCNA, DNA synthesis initiates at the origin and proceeds bidirectionally on both leading and lagging strands around the template DNA to yield duplex, circular daughter molecules. In contrast, in the absence of PCNA, early replicative intermediates containing short nascent strands accumulate. Replication forks continue bidirectionally from the origin, but surprisingly, only lagging strand products are synthesized. Thus two stages of DNA synthesis have been defined, with the second stage requiring PCNA for coordinated leading and lagging strand synthesis at the replication fork. We suggest that during eukaryotic chromosome replication there is a switch to a PCNA-dependent elongation stage that requires two distinct DNA polymerases.     

Synthesis of bacteriophage phiC DNA in dna mutants of Esherichia coli.

Host dna functions involved in the replication of microvirid phage phiC DNA were investigated in vivo. Although growth of this phage was markedly inhibited even at 35-37 degrees C even in dna+ host, conversion of the infecting single-stranded DNA into the double-stranded parental replicative form (stage I synthesis) occurred normally at 43 degrees C in dna+, dnaA, dnaB, dnaC(D), and dnaE cells. In dnaG mutant, the stage I synthesis was severely inhibited at 43 degrees C but not at 30 degrees C. The stage I replication of phiC DNA was clearly thermosensitive in dnaZ cells incubated in nutrient broth. In Tris-casamino acids-glucose medium, however, the dnaZ mutant sufficiently supported synthesis of the parental replicative form. At 43 degrees C, synthesis of the progeny replicative form DNA (stage II replication) was significantly inhibited even in dna+ cells and was nearly completely blocked in dnaB or dnaC(D) mutant. At 37 degrees C, the stage II replication proceeded normally in dna+ bacteria.     

DNA synthesis in nucleotide-permeable Escherichia coli cells. VII. Conversion of phi chi-174 DNA to its replicative form

On incubation with deoxynucleoside triphosphates and rATP, ether-treated (nucleotide-permeable) cells convert the single-stranded DNA of adsorbed bacteriophage φX174 particles to the double-stranded replicative forms. The main final product is the doubly-closed replicative form, RFI; a minor product is the relaxed form II. Interruptions in the nascent complementary strand of the viral DNA result in pieces corresponding to 5 to 10% of the unit length of the viral DNA. Pieces of similar size were previously seen in studies of the replication synthesis of Escherichia, coli DNA in ether-treated cells. Since the conversion of the single-stranded φX174 DNA to replicative form is known to be mediated entirely by host factors, it is argued that the viral single strands are replicated by macromolecular factors involed in the replication of E. coli DNA and that this is the reason why new φX174 DNA appears in short pieces. Possible consequences of this interpretation for an understanding of duplex replication are discussed. The joining of the short pieces of complementary φX174 DNA is inhibited at low deoxynucleoside triphosphate concentration (1 μM) but not by nicotinamide mononucleotide, which inhibits the NAD-dependent DNA ligase and blocks the conversion of RFII to RFI in ether-treated cells. The results are discussed with respect to previous studies on cell-DNA synthesis (Geider, 1972). It is argued that there are two polynucleotide joining mechanisms, of which only one requires NAD-dependent ligase action.     

Participation of the SSB protein in the repair of the DNA in Ehrlich ascites carcinoma cells following gamma radiation

Survival of Ehrlich ascites tumor cells, SSB content of the chromatin, and repair DNA synthesis rate were investigated after gamma-irradiation, the rate of repair synthesis was shown to depend on the SSB-protein content of the chromatin. Changes in the amount of SSB-protein in the chromatin were not connected with the postirradiation accumulation of single-strand sites in DNA.     

Studies on human DNA polymerase epsilon and GINS complex and their role in DNA replication

In eukaryotic cells, DNA replication is carried out by the coordinated action of three DNA polymerases (Pols), Pol α, δ, and ε. In this report, we describe the reconstitution of the human four-subunit Pol ε and characterization of its catalytic properties in comparison with Pol α and Pol δ. Human Pol ε holoenzyme is a monomeric complex containing stoichiometric subunit levels of p261/Pol 2, p59, p17, and p12. We show that the Pol ε p261 N-terminal catalytic domain is solely responsible for its ability to catalyze DNA synthesis. Importantly, human Pol (hPol) ε was found more processive than hPol δ in supporting proliferating cell nuclear antigen-dependent elongation of DNA chains, which is in keeping with proposed roles for hPol ε and hPol δ in the replication of leading and lagging strands, respectively. Furthermore, GINS, a component of the replicative helicase complex that is composed of Sld5, Psf1, Psf2, and Psf3, was shown to interact weakly with all three replicative DNA Pols (α, δ, and ε) and to markedly stimulate the activities of Pol α and Pol ε. In vivo studies indicated that siRNA-targeted depletion of hPol δ and/or hPol ε reduced cell cycle progression and the rate of fork progression. Under the conditions used, we noted that depletion of Pol ε had a more pronounced inhibitory effect on cellular DNA replication than depletion of Pol δ. We suggest that reduction in the level of Pol δ may be less deleterious because of its collision-and-release role in lagging strand synthesis.     

Multiplication of parvovirus LuIII in a synchronized culture system. III. Replication of viral DNA.

The replication of the single-stranded DNA (ssDNA) of parvovirus LuIII was studied in synchronized HeLa cells. After infection of the cells in early S phase, synthesis of a replicative form (RF) DNA became detectable as early as 9 h postinfection, i.e., after display of the cellular helper function(s) indispensable for the replication of LuIII virus. According to digestion with nuclease S1, hybridization studies, and electron microscopy, RF DNA is a linear, double-stranded molecule comparable in length to mature ssDNA. It sedimented around 15S in neutral solution and banded at 1.714 g/ml in CsCl. Moreover, replication of LuIII DNA obviously includes a further replicative intermediate DNA which sedimented in front of RF DNA and bore single-stranded side-chains. Newly synthesized DNA disappeared from pools containing both RF DNA and replicative intermediate DNA within 5 min and reappeared in progeny virions only after 15 min. Intranuclear accumulation of significant amounts of progeny ssDNA could not be detected. It was postulated, therefore, that newly synthesized ssDNA is immediately enclosed in a stable maturation complex and resists extraction by the method of Hirt (1967).     

Effect of Ehrlich ascites cell chalone on nascent DNA synthesis in isolated nuclei

Ehrlich Ascites Tumor (EAT) chalone has been shown to inhibit nascent DNA synthesis by inhibiting DNA polymerase alpha and beta (Nakai, 1976), but one of the problems in studying eurkaryotic DNA replication has been the relative impermeability of the cell membrane to precursors and macromolecules; hence, to circumvent this restriction without sacrificing the integrity of the replication process, a broken cell system utilizing nuclei in aqueous media was investigated. Isolated nuclei appear to continue the process of DNA replication that was proceeding in vivo before their isolation and under optimal concitions are able to initiate new synthesis (Fraser & Huberman, 1977). The effects of partially purified EAT chalone on nascent DNA could be studied directly in this nuclear system, which excluded effects of the cell membrane, nucleotide pools and other cytosol elements. A concentration-related inhibition of [3H]thymidine triphosphate ([3H]dTTP) incorporation was noted over a chalone range of 50-200 micrograms/ml. It appears that chalone can inhibit DNA polymerase alpha directly within the nucleus without an intermediate step such as a cell membrane receptor.     

Characteristics of DNA replication in isolated nuclei initiated by an aprotinin-binding protein

Isolated cell nuclei were used as the source of template DNA to investigate the role of a cytosolic aprotinin-binding protein (ADR) in the initiation of eukaryotic DNA replication. Computerized image cytometry demonstrated that the DNA content of individual nuclei increased significantly following incubation with ADR-containing preparations, and the extent of DNA synthesis is consistent with that allowed by the limiting concentration of dTTP. Thus, dTTP incorporation into isolated nuclei represents DNA synthesis and not parent strand repair. We found that dTTP incorporation into the isolated nuclei is dependent on DNA polymerase α (a principal polymerase in DNA replication) but that DNA polymerase β (a principal polymerase in DNA repair processes) does not play a significant role in this system. Finally, neither aprotinin nor a previously described cytosolic ADR inhibitor can block the replication of nuclease-treated calf thymus DNA, while both strongly inhibit replication of DNA in isolated nuclei. This result, coupled with the relative ineffectiveness of nuclease-treated DNA compared with nuclear DNA to serve as a replicative template in this assay, argues against a significant contribution from repair or synthesis which initiates at a site of DNA damage. These data indicate that ADR-mediated incorporation of ³H-dTTP into isolated nuclei results from DNA replicative processes that are directly relevant to in vivo S phase events. © 1993 Wiley-Liss, Inc.     

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.     

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms and regulation of DNA replication initiation in eukaryotes

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms.