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Biomedical subjects

P Reichard

Publications and source records attributed to P Reichard.

At least 145 records · Page 8Linked to original sources
PubMed1978

From deoxynucleotides to DNA synthesis.

The deoxyribonucleotides required for DNA synthesis are provided by reduction of ribonucleotides. Our studies aim at an understanding of the interplay between the processes involved in the production of deoxyribonucleotides and in their consumption. The enzyme system reducing ribonucleotides has been characterized in some detail, and its regulatory aspects were investigated with pure enzymes. Knowledge gained in this way made it possible to interfere specifically with the activity of ribonucleotide reductase in intact cells and to study effects on DNA synthesis. In this connection, the replication of polyoma DNA was used as a model that allowed dissection of DNA synthesis into different steps involved in the initiation and elongation processes.

Animals↗
PubMed1977

Nature of the free radical in ribonucleotide reductase from Escherichia coli.

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2. The active site of the enzyme is made up from both subunits. Protein B2 contributes inter alia an organic free radical which gives a characteristic EPR signal. This radical was now located by isotope substitution experiments to the beta position of a tyrosine residue. The EPR spectrum of protein B2 from bacteria grown in a completely deuterated medium was drastically changed. The change was reversed by the addition of other protonated amino acids. The involvement in radical formation of the beta position of tyrosine was demonstrated from EPR spectra of protein B2 from bacteria grown in the presence of specifically deuterated tyrosine.

Deuterium↗
PubMed1976

Binding of substrates to Escherichia coli ribonucleotide reductase.

Ribonucleoside diphosphate reductase from Escherichia coli consists of a 1/1 complex of two nonidentical subunits called proteins B1 and B2. The enzyme reduces the four common ribonucleoside diphosphates to the corresponding deoxyribonucleotides and is allosterically regulated by nucleoside triphosphates which influence its substrate specificity as well as its overall activity. The B1 subunit contains binding sites for the effectors while B2 contains iron and an organic free radical essential for catalytic activity. We now establish that only protein B1 binds substrates. Competition experiments support the presence of two identical substrate binding sites, distinct from the effector binding sites. The catalytic site of the enzyme thus is formed from both the B1 and B2 subunits. Dissociation constants for substrates ranged from 2 X 10(-5) to about 10(-3) M. In all cases effectors decreased these constants in agreement with their influence on the substrate specificity of ribonucleotide reductase, but did not induce cooperative effects. The increase in binding was pronounced at 20 degrees but only marginal at 0 degrees. Arrhenius plots of the influence of temperature on the catalytic activity of the enzyme showed sharp breaks at 12 degrees. The temperature effects can be interpreted as a conformational change occurring in the structure of protein B1 at the critical temperature.

Adenosine Diphosphate↗
PubMed1976

Deoxyribonucleotide pools in mouse-fibroblast cell lines with altered ribonucleotide reductase.

Mutant cells lines of 3T6 mouse fibroblasts, resistant to thymidine and deoxyadenosine, have an altered allosteric regulation of the enzyme ribonucleotide reductase (Meuth, M. and Green, H., Cell, 3, 367, 1974). Compared to 3T6, these lines contain larger pools of deoxynucleoside triphosphates, in particular deoxycytidine triphosphate, but show a normal rate of DNA synthesis. Addition of thymidine or deoxyadenosine to 3T6 cells results in large accumulations of the corresponding triphosphates and a dramatic decrease in the dCTP pool, concomitant with inhibition of DNA synthesis. Addition of thymidine to the mutant cell lines also leads to an increase in the dTTP pool but does not result in a depletion of dCTP or inhibition of DNA synthesis. Addition of deoxyadenosine only leads to a small increase of the dATP pool. In general the change in the allosteric regulation of bibonucleotide reductase is reflected in the deoxynucleotide pools.

Animals↗
PubMed1975

Replication of polyoma DNA in isolated nuclei. V. Complementation of in vitro DNA replication.

Nuclei from polyoma-infected 3T6 fibroblasts elongate in vitro the progeny strands of the replicative intermediates of polyoma DNA. When high concentrations of such nuclei were incubated, short DNA fragments were formed and subsequently added onto growing progeny strands. When nuclei were repeatedly washed with buffer containing detergent and then incubated at low concentrations. DNA synthesis was decreased. In particular, the joining process was reduced, resulting in an accumulation of short DNA fragments. All aspects of the synthetic capacity of the nuclei were restored by addition of cytoplasmic extract. Additions of purified enzymes (polynucleotide ligase from calf thymus or Escherichia coli together with E. coli DNA polymerase I) increased the joining function of the nuclei. The system can be used for the identification of the enzymatic steps concerned with polyoma DNA replication.

Animals↗
PubMed1974

Initiator RNA in discontinuous polyoma DNA synthesis.

During replication of polyoma DNA in isolated nuclei, RNA was found attached to the 5' ends of growing progeny strands. This RNA starts with either ATP or GTP and can be labeled at its 5' end with (32)P from beta-labeled nucleotides. Digestion of progeny strands with pancreatic DNase released (32)P-labeled RNA that, on gel electrophoresis, gave a distinct peak in the position expected for a decanucleotide. We believe that this short RNA is involved in the initiation of the discontinuous synthesis of DNA and propose the name "initiator RNA" for it. The covalent linkage of initiator RNA to 5' ends of growing DNA chains was substantiated by the finding that (32)P was transferred to ribonucleotides by alkaline hydrolysis of purified initiator RNA obtained by DNase digestion of polyoma progeny strands synthesized from [alpha-(32)P]dTTP. While initiator RNA was quite homogeneous in size, it had no unique base sequence since digestion with pancreatic RNase of initiator RNA labeled at its 5' end with (32)P released a variety of different [(32)P]oligonucleotides. The switch from RNA to DNA synthesis during strand elongation may thus depend on the size of initiator RNA rather than on a specific base sequence.

Adenosine Triphosphate↗
PubMed1973

RNA-linked short DNA fragments during polyoma replication.

During in vitro incubation, nuclei from polyoma-infected cells elongate the daughter strands of the replicative intermediate of polyoma DNA. This process is now shown to involve the transient formation of short fragments (4-5 S), a process that is stimulated by the addition of ribonucleoside triphosphates. The presence of stretches of RNA at the 5'-end of short DNA chains was determined from Cs(2)SO(4) equilibrium centrifugation and from the finding that isotope from alpha-(32)P-labeled deoxynucleoside triphosphates was recovered in 2'(3')-ribonucleotides after alkaline hydrolysis. Transfer occurred preferentially with [alpha-(32)P]dCTP as substrate. Starvation for deoxynucleotides by in vivo treatment with hydroxyurea resulted in the accumulation of short fragments that are deficient in RNA. Our results suggest that a late step during the discontinuous synthesis of polyoma DNA is selectively inhibited when deoxynucleotides are in short supply.

Adenosine Triphosphate↗