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At least 1,063 records · Page 59Linked to original sources
PubMed2005

Discontinuous or semi-discontinuous DNA replication in Escherichia coli?

The postulate that a stalled/collapsed replication fork will be generated when the replication complex encounters a UV-induced lesion in the template for leading-strand DNA synthesis is based on the model of semi-discontinuous DNA replication. A review of existing data indicates that the semi-discontinuous DNA replication model is supported by data from in vitro studies, while the discontinuous DNA replication model is supported by in vivo studies in Escherichia coli. Until the question of whether DNA replicates discontinuously in one or both strands is clearly resolved, any model building based on either one of the two DNA replication models should be treated with caution.

DNA Replication↗
PubMed1979

An assay system for factors involved in mammalian DNA replication.

An assay for cellular factors stimulating DNA synthesis by partially lysed CHO cells is presented. The assay is based on the observation that in highly lysed cells, DNA synthesis, as determined by [3H]dTTP incorporation, was only 2-5% of that in gently lysed cells, and that this low level of DNA synthesis could be increased by a factor of approx. 50 by the addition of CHO cell extract (i.e. supernatant of a cell homogenate subjected to high-speed centrifugation). Highly lysed cells were obtained by treatment with 0.1% Brij-58 and 240 mM KCl, while for the preparation of gently lysed cells, 0.01% Brij-58 and 80 mM KCl were used. Incorporation of [3H]dTTP reflected DNA synthesis qualitatively similar to that in intact cells. It was semiconservative, and no repair synthesis was detected unless cells were irradiated with ultraviolet light prior to parital lysis. DNA molecules of 4 S were synthesized and converted to DNA of more than 25 S via 6-12-S intermediates. DNA synthesis was restricted to nuclei from cells in S phase, and cell extract did not induce DNA synthesis in nuclei from cells in G1 phase. Stimulation of DNA synthesis by cell extract was concentration-dependent. Cell extract activity was recovered to more than 50% after (NH4)2SO4 precipitation. Heat-inactivation experiments suggested that cell extract contained at least tow factors timulating DNA replication. This system may, therefore, be used for the purification and characterization of factors participating in DNA replication of mammalian cells.

Animals↗
PubMed2003

Epigenomic stress response. Knockdown of DNA methyltransferase 1 triggers an intra-S-phase arrest of DNA replication and induction of stress response genes.

The DNA methylation pattern is an important component of the epigenome that regulates and maintains gene expression programs. In this paper, we test the hypothesis that vertebrate cells possess mechanisms protecting them from epigenomic stress similar to DNA damage checkpoints. We show that knockdown of DNMT1 (DNA methyltransferase 1) by an antisense oligonucleotide triggers an intra-S-phase arrest of DNA replication that is not observed with control oligonucleotide. The cells are arrested at different positions throughout the S-phase of the cell cycle, suggesting that this response is not specific to distinct classes of origins of replication. The intra-S-phase arrest of DNA replication is proposed to protect the genome from extensive DNA demethylation that could come about by replication in the absence of DNMT1. This protective mechanism is not induced by 5-aza-2'-deoxycytidine, a nucleoside analog that inhibits DNA methylation by trapping DNMT1 in the progressing replication fork, but does not reduce de novo synthesis of DNMT1. Our data therefore suggest that the intra-S-phase arrest is triggered by a reduction in DNMT1 and not by demethylation of DNA. DNMT1 knockdown also leads to an induction of a set of genes that are implicated in genotoxic stress response such as NF-kappaB, JunB, ATF-3, and GADD45beta (growth arrest DNA damage 45beta gene). Based on these data, we suggest that this stress response mechanism evolved to guard against buildup of DNA methylation errors and to coordinate inheritance of genomic and epigenomic information.

Cell Division↗
PubMed2004

Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly.

In mammals, heterochromatin is characterized by DNA methylation at CpG dinucleotides and methylation at lysine 9 of histone H3. It is currently unclear whether there is a coordinated transmission of these two epigenetic modifications through DNA replication. Here we show that the methyl-CpG binding protein MBD1 forms a stable complex with histone H3-K9 methylase SETDB1. Moreover, during DNA replication, MBD1 recruits SETDB1 to the large subunit of chromatin assembly factor CAF-1 to form an S phase-specific CAF-1/MBD1/SETDB1 complex that facilitates methylation of H3-K9 during replication-coupled chromatin assembly. In the absence of MBD1, H3-K9 methylation is lost at multiple genomic loci and results in activation of p53BP2 gene, normally repressed by MBD1 in HeLa cells. Our data suggest a model in which H3-K9 methylation by SETDB1 is dependent on MBD1 and is heritably maintained through DNA replication to support the formation of stable heterochromatin at methylated DNA.

Apoptosis Regulatory Proteins↗
PubMed2006

DNA primase acts as a molecular brake in DNA replication.

A hallmark feature of DNA replication is the coordination between the continuous polymerization of nucleotides on the leading strand and the discontinuous synthesis of DNA on the lagging strand. This synchronization requires a precisely timed series of enzymatic steps that control the synthesis of an RNA primer, the recycling of the lagging-strand DNA polymerase, and the production of an Okazaki fragment. Primases synthesize RNA primers at a rate that is orders of magnitude lower than the rate of DNA synthesis by the DNA polymerases at the fork. Furthermore, the recycling of the lagging-strand DNA polymerase from a finished Okazaki fragment to a new primer is inherently slower than the rate of nucleotide polymerization. Different models have been put forward to explain how these slow enzymatic steps can take place at the lagging strand without losing coordination with the continuous and fast leading-strand synthesis. Nonetheless, a clear picture remains elusive. Here we use single-molecule techniques to study the kinetics of a multiprotein replication complex from bacteriophage T7 and to characterize the effect of primase activity on fork progression. We observe the synthesis of primers on the lagging strand to cause transient pausing of the highly processive leading-strand synthesis. In the presence of both leading- and lagging-strand synthesis, we observe the formation and release of a replication loop on the lagging strand. Before loop formation, the primase acts as a molecular brake and transiently halts progression of the replication fork. This observation suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during the slow enzymatic steps on the lagging strand.

Bacteriophage T7↗
PubMed1999

DNA replication errors produced by the replicative apparatus of Escherichia coli.

It has been hard to detect forward mutations generated during DNA synthesis in vitro by replicative DNA polymerases, because of their extremely high fidelity and a high background level of pre-existing mutations in the single-stranded template DNA used. Using the oriC plasmid DNA replication in vitro system and the rpsL forward mutation assay, we examined the fidelity of DNA replication catalyzed by the replicative apparatus of Escherichia coli. Upon DNA synthesis by the fully reconstituted system, the frequency of rpsL-mutations in the product DNA was increased to 1.9x10(-4), 50-fold higher than the background level of the template DNA. Among the mutations generated in vitro, single-base frameshifts predominated and occurred with a pattern similar to those induced in mismatch-repair deficient E. coli cells, indicating that the major replication error was slippage at runs of the same nucleotide. Large deletions and other structural alterations of DNA appeared to be induced also during the action of the replicative apparatus.

Base Pairing↗
PubMed1983

DNA replication origin of polyoma virus: early proximal boundary.

We constructed a series of deleted polyoma genomes by Bal 31 nuclease digestion from the unique Bg/I site at nucleotide 86 on the "early" side of the origin of DNA replication. The ability of the cloned deleted genomes to replicate was tested after transfection into mouse 3T6 fibroblasts or into the polyomatransformed C127 (COP5) mouse cell line (Tyndall et al., Nucleic Acids Res. 9:6231-6251, 1981). Deletions up to nucleotide 64-had no effect on the amount of replicated DNA accumulated, but larger deletions, extending up to nucleotide 42, decreased this amount 7- to 10-fold. By nucleotide 38, the quantity of detected DNA was down 100-fold, and by nucleotide 20, no replication could be detected. The minimum origin segment does not contain any known high-affinity, large tumor antigen binding site.

Animals↗
PubMed1996

A protein kinase-dependent block to reinitiation of DNA replication in G2 phase in mammalian cells.

Eukaryotic cells normally replicate their DNA only once between mitoses. Unlike G1 nuclei, intact G2 nuclei do not replicate during incubation in Xenopus egg extract. However, artificial permeabilization of the nuclear membrane of G2 nuclei allows induction of new initiations by Xenopus egg extract. This is consistent with the action of a replication licensing factor which is believed to enter the nucleus when the nuclear membrane breaks down at mitosis. Here, we show that G2 nuclei will initiate a new round of replication in the absence of nuclear membrane permeabilization, if they are preexposed to protein kinase inhibitors in vivo. Competence to rereplicate is generated within 30 min of drug treatment, well before the scheduled onset of mitosis. This demonstrates that a protein kinase-dependent mechanism is continually active in G2 phase to actively prevent regeneration of replication capacity in mammalian cells. Kinase inhibition in G2 cells causes nuclear accumulation of replication protein A. Rereplication of kinase-inhibited G2 nuclei also depends on factors supplied by Xenopus egg extract, which are distinct from those required for replication licensing.

Adenine↗
PubMed1990

The effect of DNA replication on mutation of the Saccharomyces cerevisiae CDC8 gene.

Incubation in YPD medium under permissive conditions when DNA replication is going on, strongly stimulates the induction of cdc+ colonies of UV-irradiated cells of yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1). Inhibition of DNA replication by hydroxyurea, araCMP, cycloheximide or caffeine or else by incubation in phosphate buffer pH 7.0, abolishes this stimulation. Thus the replication of DNA is strongly correlated with the high induction of cdc+ colonies by UV irradiation. It is postulated that these UV-induced cdc+ colonies arise as the result infidelity in DNA replication.

Arabinonucleotides↗
PubMed2001

Inhibition of DNA replication by tirapazamine.

Tirapazamine (TPZ) is a hypoxia-selective cytotoxin that is currently being examined in Phase II and III clinical trials in combination with radiotherapy and cisplatin-based chemotherapy. Reductases convert TPZ to a cytotoxic radical that produces DNA damage under hypoxic conditions. Because one or more of the enzymes responsible for the bioactivation of TPZ is/are thought to be at or near the nuclear matrix, we hypothesized that TPZ may have a major affect on DNA replication, a process that is known to occur predominantly at the nuclear matrix. To assess the effect of TPZ on DNA replication, we measured the incorporation of radioactive thymidine into DNA of HCT116 human colon cancer cells and HeLa cells. We show that incorporation of radioactive thymidine is dramatically inhibited in cells that are pretreated with TPZ under hypoxic conditions. TPZ-induced inhibition of DNA synthesis was much greater than that produced by more toxic doses of ionizing radiation. We used the SV40-based in vitro DNA replication assay to study the mechanism of inhibition of DNA synthesis in cells treated with TPZ. Using this assay, we show that extracts prepared from cells treated with TPZ under hypoxic conditions had only 25-50% of the DNA replication activity measured in control cells. This reduction in DNA replication activity was associated with a reduction in levels of replication protein A (RPA) in cytoplasmic extracts used for the in vitro DNA replication assay and could be overcome by addition of recombinant human RPA. Furthermore, we show by indirect immunofluorescence that TPZ leads to a localization of the p34 subunit of RPA (RPA2) to small subnuclear foci. These results show that TPZ dramatically inhibits DNA replication and that the mechanism of inhibition, at least in part, involves changes in RPA that alter its cellular localization.

Antineoplastic Agents↗
PubMed1997

Early region 4 modulates adenovirus DNA replication by two genetically separable mechanisms.

Three viral proteins, all products of early region 2 (E2), participate directly in adenovirus DNA replication. Three products of early region 4 (E4) also affect viral DNA synthesis: the product of E4 ORF4 inhibits viral DNA accumulation, while the products of E4 ORFs 3 and 6 antagonize that effect of ORF4 expression. Because no E4 products are required for DNA synthesis, these proteins probably act indirectly. The E4 ORF3, 4, and 6 proteins all participate in aspects of the regulation of viral gene expression. To determine whether they modulate DNA replication by effects on expression of viral replication proteins, we examined E2 expression in E4 mutant-infected cells. In cells infected by ORF3-, 6- mutants, expression of ORF4 substantially depressed the steady-state levels of replication proteins and E2 mRNAs, reduced E2 transcription rates, and profoundly inhibited viral DNA replication. Thus, in the absence of E4 ORFs 3 and 6, ORF4 acts as a transcriptional regulator of E2 expression, and reduced replication protein levels largely account for the inhibition of DNA replication by ORF4. Cells infected by viruses that express ORFs 3 and 6 in addition to ORF4 accumulated much larger quantities of viral DNA than did cells infected by the ORF3-, 6-, 4+ mutant. Increased DNA accumulation was not accompanied by a comparable increase in E2 expression. Therefore, the ORF3 and 6 products counteract the ORF4-induced reduction of DNA replication by a mechanism other than reversing the inhibitory effect of ORF4 on E2 expression. The effect of ORF4 on E2 expression is consistent with its ability to regulate levels of the transcription factor AP-1 (Müller et al., 1992, J. Virol. 66, 5867-5878); the mechanism by which ORFs 3 and 6 enhance replication is unknown.

Adenovirus E2 Proteins↗
PubMed1984

Caffeine-induced reorganization of DNA replicating system occurs on or near nuclear matrix in HeLa cells.

Since caffeine reorganizes the DNA replicating system, with several consequences, we studied the effect of caffeine on the DNA replication which normally occurs on or near the nuclear matrix in a variety of eukaryotic cells. When HeLa cells, treated with or without the DNA-damaging agent, neocarzinostatin, were postincubated in the presence or absence of caffeine and then pulse-labeled with [3H]thymidine, the DNA remaining tightly associated with the matrix was enriched in the newly synthesized DNA at the same level as that seen in untreated cells. The nuclear matrix-bound DNA polymerase alpha activity was also the same in these cells. Therefore, in the presence of caffeine, DNA replication, with or without DNA damage, also occurs on or near the nuclear matrix, as is the case in normal DNA replication.

Caffeine↗
PubMed1976

Simian virus 40 DNA replication in nuclear monolayers.

Simian virus 40 DNA replication has been studied in nuclear monolayers prepared by treatment of monolayers of BSC-1 monkey kidney cells with Nonidet P-40. These nuclear monolayers incorporated [3H]TTP into two types of viral replicative intermediates that sediment as 25-26S and 22-23S species, respectively, in neutral sucrose gradients. The 22-23S species behaves, in dye buoyant density equilibrium gradients, as a late replicative intermediate. Examination of both species in alkaline sucrose gradients revealed the presence of two types of newly synthesized strands: (i) 4-7S strands and (ii) full-length, or nearly full-length, 10-16S strands. At low TTP concentrations (less than 0.5 muM), the two size classes were found in approximately equal amounts. However, at 10 to 50 muM TTP, the proportion of the longer strands increased, with a corresponding decrease in the relative amount of the 4-7S species. Thus, the joining of small, Okazaki-like fragments to the growing chain appears to require a much higher concentration of TTP than the synthesis of the fragments themselves. Replicating simian virus 40 DNA synthesized in the nuclear monolayers is is associated with "M bands", as previously demonstrated for replicating simian virus 40 DNA in cultured whole cells.

Cell Line↗
PubMed1977

Initiation points for DNA replication in nontransformed and simian virus 40-transformed Chinese hamster lung cells.

Randomly growing Chinese hamster lung cells were pulse-labeled with 3H-thymidine, and the replicating forks of individual DNA fibers were visualized by autoradiography. When grown in complete medium, wild-type SV40-transformed cells had more forks per unit length of DNA than nontransformed cells. In isoleucine-depleted medium, wild-type SV40-transformed cells had fewer forks per unit length than those few nontransformed cells (1-3% of the population) which continued DNA replication. Cells transformed by a tsA mutant of SV40 when grown at the permissive temperature had more forks per unit length in complete medium and fewer forks per unit length in depleted medium than nontransformed cells, but when grown at the restrictive temperature, the tsA-transformed cells behaved like nontransformed cells.

Animals↗
PubMed1986

Inhibition of DNA replication initiation by aminoglycoside antibiotics.

The reinitiation of DNA replication induced by a temperature shift in a dnaC(Ts) mutant of Escherichia coli was markedly inhibited by aminoglycoside antibiotics around the MIC in a short period. Protein synthesis continued for several minutes after the addition of aminoglycosides but was immediately blocked by chloramphenicol, suggesting that the inhibition of initiation of replication by aminoglycosides is not a secondary effect due to the interruption of protein synthesis. Aminoglycosides did not significantly affect RNA synthesis, suggesting that primer RNA synthesis for DNA initiation is not blocked by the agents. The lethal action of habekacin was observed simultaneously with the inhibition of DNA reinitiation. DNA elongation demonstrated with a dnaE(Ts) mutant or toluene-treated cells of a polA mutant was not significantly affected by aminoglycosides. The oriC-membrane complex formation was markedly interrupted by habekacin in the dnaC(Ts) mutant, and the in vitro reconstitution of the oriC-membrane complex was completely blocked by aminoglycosides. The present studies show that aminoglycosides block initiation of DNA replication and suggest that the inhibition is caused by the interruption of oriC-membrane attachment.

Aminoglycosides↗
PubMed2003

Differential DNA binding of Ku antigen determines its involvement in DNA replication.

Ku antigen (Ku70/Ku80) is a regulatory subunit of DNA-dependent protein kinase, which participates in the regulation of DNA replication and gene transcription through specific DNA sequences. In this study, we have compared the mechanism of action of Ku from A3/4, a DNA sequence that appears in mammalian origins of DNA replication, and NRE1, a transcriptional regulatory element in the long terminal repeat of mouse mammary tumor virus through which Ku antigen and its associated kinase, DNA-dependent protein kinase (DNA-PK(cs)), act to repress steroid-induced transcription. Our results indicate that replication from a minimal replication origin of ors8 is independent of DNA-PK(cs) and that Ku interacts with A3/4-like sequences and NRE1 in fundamentally different ways. UV crosslinking experiments revealed differential interactions of the Ku subunits with A3/4, NRE1, and two other proposed Ku transcriptional regulatory elements. In vitro footprinting experiments showed direct contact of Ku on A3/4 and over the region of ors8 homologous to A3/4. In vitro replication assays using ors8 templates bearing mutations in the A3/4-like sequence suggested that Ku binding to this element was necessary for replication. By contrast, in vitro replication experiments revealed that NRE1 was not involved in DNA replication. Our results establish A3/4 as a new class of Ku DNA binding site. Classification of Ku DNA binding into eight categories of interaction based on recognition and DNA crosslinking experiments is discussed.

Antigens, Nuclear↗
PubMed2001

Identification of the rhesus macaque rhadinovirus lytic origin of DNA replication.

We have identified a lytic origin of DNA replication (oriLyt) for rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in DNA replication. RRV oriLyt shares a high degree of nucleotide sequence and G+C base distribution with the corresponding loci in HHV-8.

Animals↗