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Biomedical subjects

P Reichard

Publications and source records attributed to P Reichard.

At least 127 records · Page 7Linked to original sources
PubMed1982

Effects of azidocytidine on DNA synthesis and deoxynucleotide pools of mouse fibroblast cell lines.

2'-Deoxy-2'-azidocytidine inhibits DNA synthesis of cells in tissue culture. In vitro, the diphosphate of azidocytidine was earlier shown to inhibit mammalian ribonucleotide reductase, while the triphosphate did not inhibit DNA replication in isolated cell nuclei. In intact cells, however, deoxyribonucleoside triphosphate pools were not decreased by azidocytidine in a manner expected from an inhibitor of ribonucleotide reductase. In order to differentiate between inhibition of precursor synthesis and DNA replication we studied in the present investigation the effects of azidocytidine on pool sizes of deoxynucleotides as well as on the incorporation of labeled cytidine or deoxycytidine into deoxycytidine triphosphate pools and DNA of cultured growing mouse fibroblasts. Azidocytidine was transformed to an active metabolite by phosphorylation with deoxycytidine kinase. Cell lines deficient in this enzyme showed an increased resistance against the analogue. Azidocytidine had a dual effect on the incorporation of labeled nucleosides. At low concentrations, the incorporation of cytidine into DNA was severely inhibited without a decrease of the specific activity of the dCTP pool or the size of the other three dNTP pools, suggesting a direct effect on DNA replication. At high concentrations of azidocytidine, incorporation into dCTP was also affected, demonstrating inhibition of CDP reduction. Our data also show that reduction of CDP is not necessarily tightly coupled to DNA replication since a rapid turnover of the dCTP pool occurred at the low concentrations of azidocytidine which severely inhibited DNA synthesis.

Animals↗
PubMed1981

Overproduction of the free radical of ribonucleotide reductase in hydroxyurea-resistant mouse fibroblast 3T6 cells.

Hydroxyurea inhibits the activity of ribonucleotide reductase (ribonucleoside-diphosphate reductase; 2'-deoxy-ribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) in bacteria and mammalian cells. The reductase from Escherichia coli consists of two nonidentical subunits (B1 and B2) and hydroxyurea acts by specifically destroying a tyrosine free radical of B2 required for enzyme activity. The mammalian enzyme also consists of two nonidentical subunits (M1 and M2), only one of which (M1) has been obtained in pure form. By continuous culture at stepwise increasing drug concentrations, we have now obtained a 3T6 mouse fibroblast cell line with a 100-fold increased resistance to hydroxyurea. Extracts from resistant cells showed a 3- to 15-fold increase in reductase activity. The amount of M1 protein was not increased. The amount of M2 protein could not be measured directly, but the M2 activity in extracts from resistant cells (but not normal cells) showed an EPR spectrum very similar to that of the tyrosine radical of the bacterial B2 subunit. We propose that resistance to hydroxyurea is caused either by overproduction of the complete M2 subunit or by increased generation of the tyrosine radical within the M2 protein. It seems that either alternative mirrors a possible normal regulatory mechanism for the activity of the reductase.

Animals↗
PubMed1980

Deoxyuridine triphosphate pools after polyoma virus infection.

The synthesis of polyoma DNA in virus-infected 3T6 mouse fibroblasts is discontinuous with the intermediate formation of short Okazaki fragments. Hydroxyurea, an inhibitor of the enzyme ribonucleotide reductase, inhibits polyoma DNA synthesis, as measured by incorporation of radioactive thymidine. In the inhibited state, almost all incorporation occurs into short fragments. We investigated to what extent formation of short DNA fragments might be the result of incorporation of deoxyuridine triphosphate (dUTP) into DNA, followed by excision and repair reactions. We devised a sensitive enzymatic method for measuring dUTP in cell extracts which allows the determination of the dUTP pool when this pool amounts to between 0.1 and 2% of the dTTP pool. No dUTP was detected in growing mouse fibroblasts. After infection with polyoma virus cell extracts contained 0.4% dUTP (of dTTP) at the peak of DNA synthesis. Addition of hydroxyurea at this point led to a disappearance of dUTP. We conclude that dUTP incorporation can contribute only minimally to the generation of short fragments during polyoma DNA synthesis.

Animals↗
PubMed1980

The iron center in ribonucleotide reductase from Escherichia coli.

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2. The active site is made up from both subunits. Protein B2 contains 2 iron atoms and a tyrosyl-free radical, which are essential for the enzymatic activity. The paramagnetic susceptibility of protein B2 has been measured over the temperature range 30-200 K. A deviation from the Curie law is observed at high temperatures, consistent with a structure of an antiferromagnetically coupled pair of high spin Fe(III) with an exchange coupling -J = 108(-20)+25 cm-1. Electronic spectra are resolved into components from the iron center and the radical. A band at 600 nm is clearly identified and shown to have contributions from both components. The electronic absorptions of the tyrosyl radical of protein B2 are closely similar to those reported for phenoxy radicals of tyrosine and tritertiary butyl phenol. Determinations by EPR of the amount of free radical suggest the possibility of more than one radical per active protein B2 molecule. Reconstitution of the active site from apoprotein B2 and Fe(II) is only observed in the presence of oxygen. With Fe(III), no reconstitution is obtained. The additional physical data on the iron center of protein B2 strengthen the analogy with oxidized forms of hemerythrin. The most likely structure is an antiferromagnetically coupled pair of high spin Fe(III), possibly with a bridging oxo-group.

Aerobiosis↗
PubMed1978

The tyrosine free radical in ribonucleotide reductase from Escherichia coli.

One of the two nonidentical subunits of ribonucleotide reductase from Escherichia coli, protein B2, contains an organic free radical required for enzyme activity. Earlier isotope subtitution experiments (Sjöberg, B.-M., Reichard, P. Gräslund, A., and Ehrenberg, A. (1977) J. Biol. Chem. 252, 536-541) demonstrated that the radical was localized to a tyrosine residue of the enzyme and suggested that the spin density of the radical was centered at the methylene carbon of tyrosine. However, additional isotope substitution experiments now show that the spin density of the radical must be delocalized over the aromatic ring of the tyrosine residue.

Electron Spin Resonance Spectroscopy↗
PubMed1978

Inhibition of primase, the dnaG protein of Escherichia coli by 2'-deoxy-2'-azidocytidine triphosphate.

2'-Deoxy-2'-azidocytidine-5'-triphosphate was investigated as an inhibitor in two reconstructed enzyme systems which catalyze the replication of two viral DNAs. During replication of the duplex replicative form of phiX174 DNA, DNA polymerase III holoenzyme was weakly inhibited and inhibition was reversed by dCTP. A more pronounced inhibition, not reversed by either dCTP or CTP, was observed during replication of the single-stranded DNA of the bacteriophage G4, a close relative of phiX174. This effect depended on the incorporation of 2'-deoxy-2'-azidocytidine-5'-triphosphate by primase (dnaG protein) which synthesizes a 29-residue RNA primer at the unique origin of bacteriophage G4 DNA replication. Extension of the primer strand, terminated by 2'-deoxy-2'-azidocytidine-5'-triphosphate is then severely inhibited. Primase was also inhibited by the 2'-deoxy-2'-azido derivatives of ATP, GTP, and UTP.

Coliphages↗
PubMed1978

Formation of Okazaki fragments in polyoma DNA synthesis caused by misincorporation of uracil.

When dUTP replaced dTTP during polyoma DNA replication in isolated cell nuclei, radioactivity from labeled deoxynucleoside triphosphates was almost exclusively recovered in very short Okazaki fragments and incorporation ceased after a short time. Addition of uracil, a known inhibitor of the enzyme uracil-DNA glycosidase (Lindahl et al., 1977), increased total synthesis and shifted the incorporation to longer progeny strands. The presence of as little as 2.5% of dUTP in a dTTP-containing system gave a distinct increase in isotope incorporation into Okazaki pieces accompanied by a corresponding decrease in longer strands. This effect was reversed completely by uracil. The short strands formed from dUTP could be chased efficiently into long strands. Our results suggest that dUTP can be incorporated in place of dTTP into polyoma DNA, and that polyoma-infected nuclei, similar to E. coli (Tye et al., 1977), contain an excision-repair system which by removal of uracil causes strand breakage and under certain circumstances may contribute to the formation of Okazaki fragments.

DNA, Viral↗