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G Pedrali-Noy

Publications and source records attributed to G Pedrali-Noy.

At least 19 recordsLinked to original sources
PubMed1999

Polo-like kinase 1 as a target for human cytomegalovirus pp65 lower matrix protein.

Human cytomegalovirus (HCMV) pp65 protein is the major constituent of viral dense bodies but is dispensable for viral growth in vitro. pp65 copurifies with a S/T kinase activity and has been implicated in phosphorylation of HCMV IE1 immediate-early protein and its escape from major histocompatibility complex 1 presentation. Furthermore, the presence of pp65 correlates with a virion-associated kinase activity. To clarify the role of pp65, yeast two-hybrid system (THS) screening was performed to identify pp65 cellular partners. A total of 18 out of 48 yeast clones harboring cDNAs for putative pp65 binding proteins encoded the Polo-like kinase 1 (Plk1) C-terminal domain. Plk1 behaved as a bona fide pp65 partner in THS control crosses, and the interaction was confirmed by in vitro binding experiments. Endogenous Plk1 was coimmunoprecipitated with pp65 from transiently transfected COS7 cells. In infected fibroblasts, Plk1 was coimmunoprecipitated with pp65 at late infection stages. Furthermore, Plk1 was detected within wild-type HCMV particles but not within the particles of a pp65-negative mutant (RVAd65). The hydrophilic region of pp65 was phosphorylated in vitro by Plk1. These results suggest that one function of pp65 may be to capture a cell kinase, perhaps in order to alter its activity, nucleotide preference, substrate specificity, or subcellular localization to the advantage of HCMV.

Animals↗
PubMed1997

HsN3 proteasomal subunit as a target for human immunodeficiency virus type 1 Nef protein.

HIV-1 Nef protein is important for pathogenicity, but its biochemical function remains obscure. To clarify its role, a yeast two-hybrid system (ths) screening was utilized to identify Nef cellular partners. Of 79 yeast clones harboring cDNAs for putative Nef binding proteins, 27 (34%) contained the coding region for HsN3 proteasomal subunit. HsN3 behaved as bona fide Nef partner in ths control crosses. Nef-HsN3 interaction was confirmed by in vitro binding experiments. In particular, recombinant Nef was able to capture the HsN3 subunit from a natural proteasome preparation. In Nef, the interacting region was mapped within aa 34-143, which span the structured portion of the protein, including the SH3-binding domain. In HsN3, Nef-binding portion was restricted to aa 73-249, and the tract 219-249-reminiscent of SH3 domain N-terminal 3/5ths-was shown to be essential, though not sufficient. Attempts to purify a Nef-HsN3 complex from transfected COS7 cells were unsuccessful. However, Nef was found to markedly downregulate intracellular levels of both a coexpressed HsN3 and the endogenous simian homologue. These results suggest that Nef, by binding to a subunit, might alter proteasome function in infected cells.

Amino Acid Sequence↗
PubMed1991

Purification and properties of a novel DNA methyltransferase from cultured rice cells.

DNA methyltransferase activity has been observed in a total crude homogenate of rice cells grown in suspension culture using either native plant DNA or, under the conditions used, the more responsive hemimethylated poly (dI-MedC).poly(dI-dC). Using the latter substrate we have purified an enzyme fraction 380-fold by salt extraction of chromatin, DEAE cellulose and phosphocellulose. This purified fraction showed enzyme activity only with poly (dI-MedC).poly(dI-dC) thus suggesting the occurrence in plants of a DNA methyltransferase specific for hemimethylated DNA. A Mr value of 54000 was calculated on the basis of the sedimentation coefficient which was determined by sucrose density gradient centrifugation. Apparent Km values for poly (dI-MedC).poly(dI-dC) and S-adenosyl-L-methionine were found to be 17 micrograms/ml and 2.6 microM, respectively.

Cells, Cultured↗
PubMed1990

Use of ATP, dATP and their alpha-thio derivatives to study DNA ligase adenylation.

Bacteriophage-T4 and human type I DNA ligases were found capable of self-adenylating upon exposure to both ribo- and deoxyribo-[alpha-35S]thio-ATP. However, the joining reaction does not take place in the presence of the deoxyribotriphosphates. Enzyme adenylation is reversed in all cases by an excess of PPi, but the rate of reversion is lower with thio derivatives. Therefore thio derivatives can be used to study the adenylation of DNA ligases and to search for specific inhibitors of the first step of the ligation reaction. In addition we show that thio derivatives can be used to detect DNA ligase adenylation activity covalently bound to a solid matrix.

Adenosine Triphosphate↗
PubMed1990

The effect of the minor groove binding agent DAPI (4,6-diamidino-2-phenyl-indole) on DNA-directed enzymes: an attempt to explain inhibition of plasmid expression in Escherichia coli [corrected].

The activity of DAPI, on a number of DNA-directed enzymes involved in DNA topology, transcription, replication and repair, is reported in this paper. DAPI was always more inhibitory than ethidium bromide, in particular against RNA polymerase and DNA ligase, which seemed to be specifically affected. While the effect on RNA polymerase is likely due to a preferential occupancy of the promoter region, that on DNA ligase could rely upon a mechanism of steric hindrance in the minor groove. These phenomena, independently from an alteration of the tertiary structure of DNA by the ligand, can account for the previously reported inhibition of plasmid expression in Escherichia coli.

DNA Ligases↗
PubMed1990

Effects of DNA-binding drugs on T4 DNA ligase.

A number of DNA intercalating and externally binding drugs have been found to inhibit nick sealing, cohesive and blunt end ligation, AMP-dependent DNA topoisomerization and EDTA-induced DNA nicking mediated by bacteriophage T4 DNA ligase. The inhibition seems to arise from drug-substrate interaction so that formation of active DNA-Mg2(+)-AMP-enzyme complex is impaired while assembled and active complexes are not disturbed by drug binding to the substrate.

Adenosine Monophosphate↗
PubMed1989

Flow cytometric evaluation of DNA stainability with propidium iodide after histone H1 extraction.

A flow cytometric evaluation of the effect of the histone H1 extraction on DNA stainability with propidium iodide was performed on isolated HeLa nuclei. Selective removal of the lysine-rich protein was attained by using two established techniques involving treatment with 0.7 M NaCl or low pH. DNA stainability was monitored at different dye/DNA-P ratios, varying from low to high saturating concentrations. Depletion of the histone H1from nuclei results in the transition from low to high affinity of a portion of binding sites, as shown by 1) the increase in fluorescence intensity after staining with the dye at low saturating concentrations and 2) the higher value of the fluorescence intensity ratio (FI5/FI50) exhibited by H1-depleted nuclei stained with a low (5 micrograms/ml) vs. a high (50 micrograms/ml) concentration, as compared with control samples.

Cell Nucleus↗
PubMed1989

A double-loop model for the replication of eukaryotic DNA.

Coordinated DNA synthesis of both strands at the replication fork by a fixed 'replisome' may cause dynamic and topological problems. Based upon known properties of DNA helicase, DNA primase and DNA topoisomerases, and on novel properties of DNA polymerases and DNA ligase, we propose a 'double-loop' model for the replication of eukaryotic DNA that could minimize such problems.

Animals↗
PubMed1989

Nucleoside analogs as non-substrate inhibitors of herpes simplex viruses thymidine kinase.

We have examined the capacity of a series of 6-substituted pyrimidine and 2-substituted purine derivatives to inhibit mammalian thymidine kinase and the thymidine kinases encoded by type 1 and type 2 herpes simplex viruses. Several N2-substituted guanine and deoxy guanosine derivatives displayed selective inhibitory activity against the HSV-1 and HSV-2 thymidine kinases by competing with the phosphorylation of thymidine, suggesting a possible novel pharmacological approach to herpes viruses infections.

Cells, Cultured↗
PubMed1988

Multiple roles of DNA ligase at the replication fork.

The loss of superhelical turns from a covalently closed duplex DNA exposed to bacteriophage T4 DNA ligase in the presence of AMP and Mg2+ has recently been found to be gradual and not sudden (Montecucco, A. and Ciarrocchi, G. (1988) Nucleic Acids Res. 16, 7369-7381). In this paper, we show that the AMP-dependent DNA relaxation catalyzed by human and E. coli DNA ligases also takes place according to a step-wise mechanism. DNA relaxation is inhibited by pyrophosphate, by ATP (or NAD in the case of the E. coli enzyme) and by high ionic strength and is essentially distributive with the human or T4 DNA ligases, and processive with the bacterial one. The AMP-dependent ability of DNA ligases to relax DNA might allow these enzymes to relieve possible topological complications of the nascent double helix generated by the replication of the lagging strand.

Adenosine Monophosphate↗
PubMed1988

DNA unwinding and inhibition of T4 DNA ligase by anthracyclines.

The ability to alter DNA tertiary structure of ten anthracycline derivatives whose antitumor potency is known was studied by an assay that makes use of nicked circular DNA and bacteriophage T4 DNA ligase. This assay allows the detection of tertiary structure alterations caused by DNA binding of both intercalating and non-intercalating drugs. The determination of these events can be obtained at different temperatures in the range of activity of DNA ligase. The results indicate that anthracyclines alter the DNA tertiary structure but this property does not correlate with their cytotoxic or antitumor activities. An additional interesting finding was that several anthracyclines inhibit T4 DNA ligase. The inhibition can be complete and is a cubic function of drug concentration. The inhibition of DNA ligase does not correlate with the ability of anthracyclines to alter the tertiary structure of DNA but is dependent from the presence of an amino group on the sugar ring.

Antibiotics, Antineoplastic↗
PubMed1987

Alteration of DNA tertiary structure by physical and chemical carcinogens: involvement in DNA repair processes.

Parameters defining the topological state of DNA seem extremely important for describing the reactive state of the same DNA molecules. We have shown that physical and chemical DNA modifying agents alter the tertiary structure of DNA molecules. Variations in the tertiary structure of DNA were studied by one dimensional electrophoresis on an agarose gel of partially relaxed plasmid DNA topoisomers, a technique allowing the measurement of alterations in the degree of supercoiling equivalent to fractions of superhelical turns. Unwinding angles of -10.1 degrees or -8.7 degrees per pyrimidine or thymine dimer respectively, of -12 degrees per apurinic site, and of -3.4 degrees per methylated purine were obtained by titrating the number of damaged sites necessary to reduce the number of superhelical turns by one in each topoisomer. On the contrary, enzymatic methylation of the C-5 position of cytosine (a modified base present in prokaryotic and eukaryotic DNAs) did not alter the DNA tertiary structure. We have also shown that local alterations in DNA structure caused by UV-irradiation inhibit bacterial DNA topoisomerase I and DNA methylase, and that the topological state of DNA substrate influences the mode of methylation of Hpa II DNA methylase. These findings suggest that the natural topological state of DNA substrate (linear, relaxed, or covalently closed duplex DNA with varying degrees of supercoiling) influences the mode of action of enzymes possibly involved in DNA repair processes, while DNA structural alterations caused by DNA modifying agents might influence DNA repair processes in two ways: either by driving the interaction between repair enzymes and the modified sites of DNA, or by inhibiting or changing the mode of action of enzymes normally acting on unmodified DNA.

Animals↗
PubMed1986

Mammalian DNA methyltransferases prefer poly(dI-dC) as substrate.

The synthetic duplex DNA, poly(dI-dC).poly(dI-dC), is methylated in vitro by human or murine DNA methyltransferases at 20-100 times the rate of other nonmethylated DNAs. Preparation of the hemimethylated derivative, poly(dI-dMeC).poly(dI-dC), of this polymer increases its effectiveness as a substrate by 2-fold, making it 4-10 times more effective as a substrate for mammalian DNA methyltransferases than any other hemimethylated DNA so far reported. However, the apparent slower rate of de novo methylation of poly(dI-dC).poly(dI-dC) as compared to the hemimethylated derivative is due to substrate inhibition, unique to the unmethylated polymer, as the rates of de novo and maintenance methylation are identical at low substrate concentrations.

Animals↗
PubMed1985

DNA methyltransferases: activity minigel analysis and determination with DNA covalently bound to a solid matrix.

We describe two methods that facilitate detection and characterization of DNA methyltransferases: activity gel analysis and the use of DNA-cellulose or DNA-Sepharose in DNA methylation reactions. The first permits identification of catalytic subunits, determination of the influence of proteolysis, and evolutionary or developmental studies. The second allows accurate and fast determination of DNA methyltransferase activities in crude extracts and during purification.

Catalysis↗