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At least 541 records · Page 30Linked to original sources
PubMed1974

Endonuclease activity associated with purified PM2 bacteriophages.

An endonucleolytic activity is associated with purified PM(2) bacteriophages. It converts the double-stranded supercoiled PM(2) DNA mostly into the linear form that sediments as a homogenous peak in alkaline sucrose gradients. The same activity is found in intact or detergent-lysed phages. Optimal activity is observed between pH 6.8 and 7.5 at 28 degrees . Divalent cations, Mg(2+) or Mn(2+), are necessary for activity, and the enzyme is inhibited by RNA. The endonuclease has no appreciable activity on linear DNA molecules, but it attacks single-stranded circular DNA.

Bacteriophages↗
PubMed2000

The p53 tumor suppressor stimulates the catalytic activity of human topoisomerase IIalpha by enhancing the rate of ATP hydrolysis.

DNA topoisomerase II is an essential nuclear enzyme for proliferation of eukaryotic cells and plays important roles in many aspects of DNA processes. In this report, we have demonstrated that the catalytic activity of topoisomerase IIalpha, as measured by decatenation of kinetoplast DNA and by relaxation of negatively supercoiled DNA, was stimulated approximately 2-3-fold by the tumor suppressor p53 protein. In order to determine the mechanism by which p53 activates the enzyme, the effects of p53 on the topoisomerase IIalpha-mediated DNA cleavage/religation equilibrium were assessed using the prototypical topoisomerase II poison, etoposide. p53 had no effect on the ability of the enzyme to make double-stranded DNA break and religate linear DNA, indicating that the stimulation of the enzyme catalytic activity by p53 was not due to alteration in the formation of covalent cleavable complexes formed between topoisomerase IIalpha and DNA. The effects of p53 on the catalytic inhibition of topoisomerase IIalpha were examined using a specific catalytic inhibitor, ICRF-193, which blocks the ATP hydrolysis step of the enzyme catalytic cycle. Clearly manifested in decatenation and relaxation assays, p53 reduced the catalytic inhibition of topoisomerase IIalpha by ICRF-193. ATP hydrolysis assays revealed that the ATPase activity of topoisomerase IIalpha was specifically enhanced by p53. Immunoprecipitation experiments revealed that p53 physically interacts with topoisomerase IIalpha to form molecular complexes without a double-stranded DNA intermediary in vitro. To investigate whether p53 stimulates the catalytic activity of topoisomerase II in vivo, we expressed wild-type and mutant p53 in Saos-2 osteosarcoma cells lacking functional p53. Wild-type, but not mutant, p53 stimulated topoisomerase II activity in nuclear extract from these transfected cells. Our data propose a new role for p53 to modulate the catalytic activity of topoisomerase IIalpha. Taken together, we suggest that the p53-mediated response of the cell cycle to DNA damage may involve activation of topoisomerase IIalpha.

Adenosine Triphosphate↗
PubMed2004

Bacteriophage T4 32 protein is required for helicase-dependent leading strand synthesis when the helicase is loaded by the T4 59 helicase-loading protein.

In the bacteriophage T4 DNA replication system, T4 gene 59 protein binds preferentially to fork DNA and accelerates the loading of the T4 41 helicase. 59 protein also binds the T4 32 single-stranded DNA-binding protein that coats the lagging strand template. Here we explore the function of the strong affinity between the 32 and 59 proteins at the replication fork. We show that, in contrast to the 59 helicase loader, 32 protein does not bind forked DNA more tightly than linear DNA. 32 protein displays a strong binding polarity on fork DNA, binding with much higher affinity to the 5' single-stranded lagging strand template arm of a model fork, than to the 3' single-stranded leading strand arm. 59 protein promotes the binding of 32 protein on forks too short for cooperative binding by 32 protein. We show that 32 protein is required for helicase-dependent leading strand DNA synthesis when the helicase is loaded by 59 protein. However, 32 protein is not required for leading strand synthesis when helicase is loaded, less efficiently, without 59 protein. Leading strand synthesis by wild type T4 polymerase is strongly inhibited when 59 protein is present without 32 protein. Because 59 protein can load the helicase on forks without 32 protein, our results are best explained by a model in which 59 helicase loader at the fork prevents the coupling of the leading strand polymerase and the helicase, unless the position of 59 protein is shifted by its association with 32 protein.

Bacteriophage T4↗
PubMed1985

Osmotic pressure of DNA solutions and effective diameter of the double helix.

A simple osmometer with nuclear filters (polymer films with pores of a preset diameter) were used to measure the osmotic pressure of Col E1 plasmid DNA solutions in the concentration range of 1-4 mg/ml DNA. Linear and open circular DNA forms proved to have the same osmotic pressure within the experimental accuracy. The results of the measurements were used for calculating the second virial coefficient A2 of the solution of DNA segments and the effective chain diameter d eff in the ionic strength range of 10(-2)-0.1 M. As the ionic strength is lowered from 0.1 to 10(-2) M the effective diameter of DNA increases from 80 to 220 A. The results are in rather good agreement with theory and with other experimental data.

DNA, Bacterial↗
PubMed1986

On the compact form of linear duplex DNA: globular states of the uniform elastic (persistent) macromolecule.

A theory of collapse of DNA considered as unifilar homopolymer is suggested. The collapse is interpreted as the coil-globule transition. Three reasons of the collapse such as the confinement in a microcavity, the influence of poor low-molecular-weight solvent and the influence of polymeric solvent were studied. The results are summed up by the stage diagrams in variables: DNA length versus the characteristics of the compaction factor (the cavity volume, the energy of attraction of DNA segments in poor low-molecular-weight solvent and the concentration of polymer added). It is shown that a sufficiently long DNA forms the spherical compact particle while the relatively short DNA forms the toroidal one. More delicate features of the tertiary structure are determined by the relative role of the bending stiffness and steric repulsions in preventing further collapse. As the compaction occurs in polymeric solvent almost all added polymer is forced out from the globule. Thus, the internal structure of the compact DNA particle in polymeric solvent is similar to that in the model of microcavity.

DNA↗
PubMed2000

The effects of microgravity on ligase activity in the repair of DNA double-strand breaks.

PURPOSE: In recent years, contradictory data have been reported about the effects of microgravity on radiation-induced biological responses in space experiments. The aim of the present study was to clarify whether enzymatic repair of DNA double-strand breaks is affected by microgravity using an in vitro enzymatic reaction system. MATERIALS AND METHODS: The DNA repair activity of T4 DNA ligase (EC 6.5.1.1) was measured in vitro for a DNA substrate damaged by restriction enzyme digestion during a US Space Shuttle mission (Discovery; STS-91). After the flight, the amount of ligated DNA molecules was measured using an electrophoresis method. RESULTS: Ligated products (closed circular DNA, open circular DNA and multimeric ligated products) were produced by T4 DNA ligase treatment of linear DNA containing double-strand breaks, and they increased with increasing T4 DNA ligase concentration (0-3 units per microg of plasmid DNA). Almost no difference in T4 DNA ligase activity was detected between the space experiments and the control ground experiments. CONCLUSIONS: No significant effect of microgravity on ligation of damaged DNA was found during space flight. Therefore, other mechanisms must account for the synergism between radiation and microgravity, if it exists.

DNA Damage↗
PubMed1991

Visualization of novel simian virus 40 DNA recombination intermediates induced by ultraviolet light irradiation.

Electron microscopic technique was used to examine the structures of SV40 DNA recombination intermediates induced by ultraviolet irradiation as an approach for understanding recombination mechanisms in animal cells. Putative recombination intermediate with the characteristic Holliday junction was observed in both SV40 and CV-1 monkey kidney cell DNA. These results suggest that Holliday recombination intermediate is a common intermediate in eukaryotic as well as prokaryotic recombination pathways. In UV irradiated cells, putative SV40 DNA recombination intermediates with multiple recombining partners were observed. In addition, UV irradiation induced two types of novel joint molecules of SV40 DNA. The first type contains replication intermediates as one of the joint molecules with the putative recombination junction located in the newly replicated DNA arms. The second type of novel joint molecules is represented by of the 'dumbbell' structures with two circular SV40 DNA linked by a linear DNA of varying lengths. The structures of these novel recombination intermediates suggest a strand-invasion mechanism for UV-induced DNA recombination.

Animals↗
PubMed1995

Interaction of normal and mutant SRY proteins with DNA.

In mammals, sex determination is caused by the Y-chromosome gene SRY. The DNA-binding domain of human SRY protein is similar to those of the chromatin protein HMG1. Like HMG1, SRY binds to kinked DNA structures, and bends linear DNA sharply upon binding. We analysed the biochemical properties of mutant SRY proteins from five patients with complete gonadal dysgenesis: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally, and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence, thus discriminating different target sites. We suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY in chromatin is essential for the expression of genes involved in testis differentiation.

Base Sequence↗
PubMed1982

Kinetics of protein and DNA synthesis studied by mathematical modelling of flow cytometric protein and DNA histograms.

Mathematical models for histograms of cellular protein content as measured by flow cytometry were developed, based on theoretical protein distributions. These were derived from the age distribution of cells and the accumulation function for cellular protein content as a function of age within the cell cycle. A model assuming an exponential age distribution and an exponential protein accumulation function was found to give the best representation of protein histograms of exponentially growing NHIK 3025 cells. This is in good agreement with the known kinetic behaviour of such cells. By the combined use of the protein histogram model and a similar model for DNA content, and assuming linear DNA accumulation during S, the fraction of cells in S, as a function of cellular protein content, was simulated. This function showed good agreement with values of the [3H]TdR labelling index scored in cells sorted by flow cytometry from 5-channel intervals of the protein histogram. The protein and DNA histogram models were combined into a two-dimensional model for correlated protein/DNA measurements. Comparison between simulated data and experimentally derived two-dimensional protein/DNA histograms gave further support to the cell kinetic assumptions underlying the models, but also identified some minor deviations which could not be recognized in the analysis of the one-dimensional histograms.

Cell Cycle↗
PubMed1995

The relaxation of supercoiled DNA molecules as a biophysical dosimeter for ionizing radiations: a feasibility study.

In this paper we explore the feasibility of using DNA molecules as a biophysical radiation dosimeter. Supercoiled phi X174 bacteriophage DNA molecules were irradiated with different gamma radiation doses. The strand breakage produced by ionizing radiation within supercoiled double-stranded DNA molecules (RFI) yields relaxed circular DNA molecules (RFII) and linear DNA molecules (RFIII) as a result of single-strand breaks and double-strand breaks, respectively. The irradiated samples were subjected to electrophoresis on agarose gels to separate the three forms. A proprietary fluorescent dye was used to detect DNA bands within the gel, which was photographed under UV transillumination. The negative was scanned with a computerized imaging densitometric system for DNA band quantitation. The relative fractions of the three molecular forms are dose dependent, and can be modeled mathematically with five parameters. The values of the parameters were determined by optimizing the fit of the model to the data, using a nonlinear regression procedure of a commercial statistical analysis package. Once the parameters of DNA breakage have been determined, absorbed dose can be measured by this technique, which we have termed supercoil relaxation dosimetry. The average accuracy of dose determination for our system over the range of 1-40 Gy was about 5%. Supercoil relaxation dosimetry may be well suited to certain difficult dosimetric problems.

Algorithms↗
PubMed1984

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.

Base Sequence↗
PubMed2006

High-mobility-group box nuclear factors of Plasmodium falciparum.

In eukaryotes, the high-mobility-group (HMG) nuclear factors are highly conserved throughout evolution and are divided into three families, including HGMB, characterized by an HMG box domain. Some HMGB factors are DNA structure specific and preferentially interact with distorted DNA sequences, trigger DNA bending, and hence facilitate the binding of nucleoprotein complexes that in turn activate or repress transcription. In Plasmodium falciparum, two HMGB factors were predicted: PfHMGB1 and PfHMGB2. They are small proteins, under 100 amino acids long, encompassing a characteristic HMG box domain closely related to box B of metazoan factors, which comprises two HMG box domains, A and B, in tandem. Computational analyses supported the conclusion that the Plasmodium proteins were genuine architectural HMGB factors, and in vitro analyses performed with both recombinant proteins established that they were able to interact with distorted DNA structures and bend linear DNA with different affinities. These proteins were detected in both asexual- and gametocyte-stage cells in Western blotting experiments and mainly in the parasite nuclei. PfHMGB1 is preferentially expressed in asexual erythrocytic stages and PfHMGB2 in gametocytes, in good correlation with transcript levels of expression. Finally, immunofluorescence studies revealed differential subcellular localizations: both factors were observed in the nucleus of asexual- and sexual-stage cells, and PfHMGB2 was also detected in the cytoplasm of gametocytes. In conclusion, in light of differences in their levels of expression, subcellular localizations, and capacities for binding and bending DNA, these factors are likely to play nonredundant roles in transcriptional regulation of Plasmodium development in erythrocytes.

Amino Acid Sequence↗
PubMed1976

Construction and analysis of viable deletion mutants of simian virus 40.

Viable mutants of simian virus 40 (SV40), with deletions ranging in size from 15 to 200 base pairs, have been obtained by infecting CV-1P cells with circularly permuted linear SV40 DNA. The linear DNA was produced by cleavage of closed circular DNA with DNase I in the presence of Mn2+, followed, in some cases, by mild digestion with lambda 5'-exonuclease. The SV40 map location and the size of each deletion were determined by using the S1 nuclease mapping procedure (Shenk et al., 1975) and the change in size of fragments produced by Hind II + III endonuclease cleavage. Deletions in at least three regions of the SV40 chromosome have slight or no effect on the rate or yield of viral multiplication and on vira-induced cellular transformation. These regions are located at the following coordinates on the SV40 physical map: 0.17 to 0.18; 0.54 to 0.59; and 0.68 to 0.74.

Cell Line↗
PubMed1988

Electroporation-mediated transfection of Acholeplasma laidlawii with mycoplasma virus L1 and L3 DNA.

In contrast to mycoplasma virus L1 and L2 circular DNA, mycoplasma virus L3 linear DNA is not biologically active in polyethylene glycol-mediated transfection. Electroporation of Acholeplasma laidlawii, however, leads to plaque formation after incubation with L3 DNA. The efficiency of electroporation-mediated transfection is 1/10 that of polyethylene glycol-mediated transfection as estimated with L1 DNA. Trypsin treatment of cells before DNA addition increases the efficiency of DNA uptake.

Acholeplasma laidlawii↗