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At least 1,729 records · Page 96Linked to original sources
PubMed1985

Relations between synthesis of deoxyribonucleotides and DNA replication in 3T6 fibroblasts.

In exponentially growing 3T6 cells, the synthesis of deoxythymidine triphosphate (dTTP) is balanced by its utilization for DNA replication, with a turnover of the dTTP pool of around 5 min. We now investigate the effects of two inhibitors of DNA synthesis (aphidicolin and hydroxyurea) on the synthesis and degradation of pyrimidine deoxynucleoside triphosphates (dNTPs). Complete inhibition of DNA replication with aphidicolin did not decrease the turnover of pyrimidine dNTP pools labeled from the corresponding [3H]deoxynucleosides, only partially inhibited the in situ activity of thymidylate synthetase and resulted in excretion into the medium of thymidine derived from breakdown of dTTP synthesized de novo. These data demonstrate continued synthesis of dTTP in the absence of DNA replication. In contrast, hydroxyurea decreased the turnover of pyrimidine dNTP pools 5-50-fold. Hydroxyurea is an inhibitor of ribonucleotide reductase and stops DNA synthesis by depleting cells of purine dNTPs but not pyrimidine dNTPs. Our results suggest that degradation of dNTPs is turned off by an unknown mechanism when de novo synthesis is blocked.

Animals↗
PubMed2005

Repression of DNA replication licensing in quiescence is independent of geminin and may define the cell cycle state of progenitor cells.

The DNA replication (or origin) licensing machinery ensures precise duplication of the genome and contributes to the regulation of proliferative capacity in metazoa. Using an in vitro fibroblast model system coupled to a cell-free DNA replication assay, we have studied regulation of the origin licensing pathway during exit from and re-entry into the mitotic cell cycle. We show that in the quiescent state (G0) loss of proliferative capacity is achieved in part through down-regulation of the replication licensing factors Cdc6 and Mcm2-7. The origin licensing repressor geminin is absent in quiescent fibroblasts, suggesting that this powerful inhibitor of the licensing machinery is not required to suppress proliferative capacity in G0. Geminin expression is induced at a late stage in the G0-S transition post pre-RC assembly. Ectopic geminin can block re-acquisition of DNA replication competence during re-entry into the cell cycle, indicating that geminin levels must be tightly down-regulated for escape from G0. Analysis of geminin levels in thyroid shows that geminin expression is suppressed in anatomical compartments/tissues harbouring quiescent cells, confirming our in vitro data. Spatio-temporal control of geminin expression may therefore be of particular relevance for multi-potential stem cells which cycle infrequently. In support of this hypothesis, we have identified a unique population of cells in the putative stem cell niche of intestinal epithelium that are unlicensed and lack geminin expression, a prerequisite for successful re-entry into cycle. Our data argue that the prolonged cell cycle times observed for intestinal stem cells could be due to exit of progenitor cells from cycle into an unlicensed "out-of-cycle" state, a powerful mechanism by which rapidly proliferating tissues may resist genotoxic insult.

3T3 Cells↗
PubMed2006

The control mechanism for lagging strand polymerase recycling during bacteriophage T4 DNA replication.

Given the polarity of DNA duplex, replication by the leading strand polymerase is continuous whereas that by the lagging strand polymerase is discontinuous proceeding through Okazaki fragments. Yet the respective polymerases act processively, implying that the recycling of the lagging strand polymerase is a controlled process. We demonstrate that the rate of the lagging strand polymerase relative to that of fork movement affects Okazaki fragment size and generates ssDNA gaps. We show by using a substrate with limited priming sites that Okazaki fragments can be shifted to shorter lengths by varying the rate of the primase. We find that clamp and clamp loader levels affect both primer utilization and Okazaki fragment size, possibly implicating clamp loading onto the RNA primer in the mechanism of lagging strand polymerase recycling. We formulate a signaling model capable of rationalizing the distribution of Okazaki fragments under various conditions for this and possibly other replisomes.

Bacteriophage T4↗
PubMed2005

Cdc7-Drf1 is a developmentally regulated protein kinase required for the initiation of vertebrate DNA replication.

Cdc7, a protein kinase required for the initiation of eukaryotic DNA replication, is activated by a regulatory subunit, Dbf4. A second activator of Cdc7 called Drf1 exists in vertebrates, but its function is unknown. Here, we report that in Xenopus egg extracts, Cdc7-Drf1 is far more abundant than Cdc7-Dbf4, and removal of Drf1 but not Dbf4 severely inhibits phosphorylation of Mcm4 and DNA replication. After gastrulation, when the cell cycle acquires somatic characteristics, Drf1 levels decline sharply and Cdc7-Dbf4 becomes the more abundant kinase. These results identify Drf1 as a developmentally regulated, essential activator of Cdc7 in Xenopus.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Role of yeast Rth1 nuclease and its homologs in mutation avoidance, DNA repair, and DNA replication.

The RTH1(RAD27) gene of Saccharomyces cerevisiae encodes a structure-specific endonuclease that cleaves 5'-ended single-stranded DNA at its junction with duplex DNA. Genetic and biochemical studies have indicated a role of Rth1 nuclease in the removal of RNA primers formed during DNA replication. The rth1Delta mutation confers temperature-sensitive lethality, and increases sensitivity to alkylating agents. The instability of repetitive DNA is greatly enhanced in the rth1Delta mutant. The conditional lethality of the rth1Delta mutation indicates that another nuclease can function in DNA replication in the absence of RTH1. RAD2, a homolog of RTH1, is required for nucleotide-excision repair. Here, we examine three other homologs of RTH1/RAD2 - YEN1, EXO1, and DIN7. Deletion of any of these genes in the rth1Delta strain has no effect on cell viability, suggesting the involvement of another, and as yet unidentified, nuclease in the maturation of Okazaki fragments. Our data also indicate that only RTH1 functions in the repair of alkylation damage. Deletions of YEN1, EXO1, DIN7, or RAD2, either singly or when combined with one another and with the rth1Delta mutation, have no effect on the rate of instability of dinucleotide repeats or on the rate of formation of large duplications in the CAN1 gene. These data provide evidence of a high degree of specificity for the role of RTH1 in DNA replication and in base-excision repair, and for the requirement of RAD2 in nucleotide-excision repair. The possibility that both Rth1 and Exo1 function in DNA mismatch repair is discussed.

Alkylation↗
PubMed1976

Late events in T4 bacteriophage production. I. Late DNA replication is primarily exponential.

The possibility of a switch in the mechanism of T4 DNA replication, from an exponential-bidirectional mode at early stages to a nonexponential (rolling circle) mode at later stages of phage development, has been investigated. The conclusion that DNA replication does not involve such a change in mechanism for the majority of replicating molecules is based on the analysis of the clonal distribution of mutants specifically induced at late times after infection. The clonal distribution of mutants, induced by adding 5-bromodeoxyuridine to infected cells at a time when 100 phage equivalents of DNA had accumulated, fits the pattern predicted by exponential replication.

Bromodeoxyuridine↗
PubMed1995

Appearance of rereplication and activation of DNA replication by calyculin A in a cell-free extract of activated Xenopus eggs.

When sperm nuclei were added to Xenopus S phase (activated) egg extracts pretreated with a serine/threonine protein phosphatase inhibitor, calyculin A, a density substitution experiment showed that newly synthesized sperm DNA migrated in two peaks, heavy-light DNA and heavy-heavy DNA, only in the presence of calyculin A and that the total DNA replication activity was activated. In contrast, the addition of calyculin A to S phase extracts about 30 min after addition of sperm nuclei had no effect on DNA replication activity. Calyculin A clearly prevented the decline of Replication Licensing Factor activity during S phase. These results imply that the occurrence of some rereplication and the activation of DNA replication by calyculin A in activated extracts may be due to the inhibition of inactivation of licensed sites of initiation or the increase in the number of the initiation sites of DNA replication because of lack of the fall in Licensing Factor activity during S phase.

Animals↗
PubMed1995

Cdc54 belongs to the Cdc46/Mcm3 family of proteins which are essential for initiation of eukaryotic DNA replication.

CDC54 is a gene essential for initiation of DNA replication in Saccharomyces cerevisiae, and which is known to genetically interact with other regulators of the S-phase, including CDC46. We describe the isolation and sequencing of CDC54 and show that it encodes a protein structurally related to Cdc46p, Mcm2p and Mcm3p by the presence of a conserved domain of 145 amino acids which is internal to each polypeptide. This conserved domain resembles the DEAD box of RNA helicases and is similar to the conserved domain associated with a group of transcription and replication factors with known or assumed DNA-dependent ATPase activity, suggesting it may be involved in nucleic-acid recognition. Comparison of Cdc54p to related proteins from other species revealed that it closely resembles cdc21p from Schizosaccharomyces pombe.

Amino Acid Sequence↗
PubMed2001

Inhibition of hepatitis B virus DNA replication by imino sugars without the inhibition of the DNA polymerase: therapeutic implications.

Previously we have shown that the imino sugar inhibitor of N-linked glycan processing, N-nonyl-deoxynojirimycin (N-nonyl-DNJ), had antiviral activity in the woodchuck model of chronic hepatitis B virus (HBV) infection. In studying the mechanism of action of this compound, it was discovered that imino sugars could inhibit HBV secretion without inhibiting N-linked glycoprocessing. Although N-nonyl-DNJ is an inhibitor of the endoplasmic reticulum (ER) glucosidase, here it is shown that N-nonyl-DNJ retained antiviral activity at concentrations that had no significant impact on ER glucosidase function. Taken together, these results suggested that N-nonyl-DNJ possessed an antiviral activity attributable to a function other than an impact on glycoprocessing. This hypothesis was confirmed by experiments showing that N-nonyl-deoxygalactojirimycin (N-nonyl-DGJ), an alkyl derivative of galactose with no impact on glycoprocessing, retains anti-HBV activity. The data suggest that N-nonyl-DGJ exerts its antiviral action at a point before viral envelopment and may prevent the proper encapsidation of the HBV pregenomic RNA.

1-Deoxynojirimycin↗
PubMed1980

DNA replication of bacteriophage T5. 1. Fractionation of intracellular T5 DNA by agarose gel electrophoresis.

Two forms of DNA may be isolated from gently lysed T5-infected bacteria. In neutral sucrose gradients a fast sedimenting form (fsf) is distinguishable from a slow sedimenting form (ssf) which moves at a rate similar to that of DNA extracted from mature T5 virus particles. A method of agarose gel electrophoresis is described which gives complete separation of the slow sedimenting from the fast sedimenting form. The DNA was extracted from the agarose using potassium iodide and isopycnic centrifugation; samples of the slow sedimenting form prepared by these methods were suitable for detailed structural studies.

Centrifugation, Density Gradient↗
PubMed1977

Regulation of deoxyribonucleotide biosynthesis during in vivo bacteriophage T4 DNA replication. Intrinsic control of synthesis of thymine and 5-hydroxymethylcytosine deoxyribonucleotides at precise ratio found in DNA.

The kinetics of the de novo formation of pyrimidine deoxyribonucleotides is the same after infection by wild type bacteriophage T4, which generate very low steady state levels of deoxytibonucleotides, and by T4 DNA synthesis-negative mutatants (Dna-), which accumulate high levels, suggesting that the control is not by a feedback mechanism. In this study, the ratio of the de novo synthesis of dTMP to HmdCMP derivatives was measured by determining the total thymine and 5-hydroxylxytosine (HmCyt) deoxyribonucleotides synthesized by the reductive pathways from [6-3H]uracil including those in DNA and any degradation products excreted into the medium. The ratio of the de novo synthesis of Thy/HmCyt derivatives remained constant at 2.1 +/- 0.1 for at least 45 min after infection by wild type phage, i.e. precisely at the Thy/HmCyt ratio in T4 DNA. On infection by phage mutated in the Dna-genes 32, 41, 44, or 45, the ratio still remained close to 2 to 1 for at least 25 min. Only after the pyrimidine deoxyribonucleotide concentrations reached levels about 100-fold greater than the initial values did the ratio begin to increase. However, a mutant of the structural gene for T4 DNA polymerase showed some increase in ratio by 15 min. Mutants of gene 1 (HmdCMP kinase) were distinct in that the Thy/HmCyt ratio dropped to about 1.0 by 25 min, and then remained quite constant. Uniquely, in these mutants a significant quantity of 5-hydroxymethyluracil or a derivative was found, about 40% being in the medium. The product was shown to be derived by deamination of a 5-HmCyt derivative. All Dna- mutants tested excreted 35 to 50% of their thymine derivatives, mostly as thymine, into the medium. Neither thymine nor 5-hydroxymethyluracil derivates were excreted after wild type phage infection. We propose that pyrimidine deoxyribonucleotide synthesis is regulated at a Thy:HmCyt ratio of 2:1 as an intrinsic property of a complex of enzymes synthesizing and channeling deoxyribonucleotides for T4 DNA replication and not exclusively by effector-sensitive mechanisms.

Coliphages↗