Lars Thelander

Continual presence of oxygen and iron required for mammalian ribonucleotide reduction: Possible regulation mechanism

A radical-free preparation of a highly purified ribonucleotide reductase from calf thymus was shown to generate an M2-specific tyrosine free radical on incubation with iron and dithiothreitol in the presence of air. The radical is essential for activity but once formed has a half-life of about 10 min. Using the calf thymus enzyme, there is a continual requirement of oxygen and iron for ribonucleotide reduction indicating a continual regeneration of the radical during enzyme catalysis. We therefore propose that one way a cell may regulate ribonucleotide reductase activity is by controlling the generation of M2-specific tyrosine free radicals within existing M2 molecules.

Cross-talk between the Allosteric Effector-binding Sites in Mouse Ribonucleotide Reductase

We compared the allosteric regulation and effector binding properties of wild type R1 protein and R1 protein with a mutation in the “activity site” (D57N) of mouse ribonucleotide reductase. Wild type R1 had two effector-binding sites per polypeptide chain: one site (activity site) for dATP and ATP, with dATP-inhibiting and ATP-stimulating catalytic activity; and a second site (specificity site) for dATP, ATP, dTTP, and dGTP, directing substrate specificity. Binding of dATP to the specificity site had a 20-fold higher affinity than to the activity site. In all these respects, mouse R1 resemblesEscherichia coli R1. Results with D57N were complicated by the instability of the protein, but two major changes were apparent. First, enzyme activity was stimulated by both dATP and ATP, suggesting that D57N no longer distinguished between the two nucleotides. Second, the two binding sites for dATP both had the same low affinity for the nucleotide, similar to that of the activity site of wild type R1. Thus the mutation in the activity site had decreased the affinity for dATP at the specificity site, demonstrating the interaction between the two sites.

Altered expression of ribonucleotide reductase and role ofM2 gene amplification in hydroxyurea-resistant hamster, mouse, rat, and human cell lines

Five hamster, mouse, and rat cell lines resistant to the cytotoxic effects of hydroxyurea have been characterized. All cell lines contained increased ribonucleotide reductase activity, elevated levels of the M2 component of ribonucleotide reductase as judged by electron paramagnetic resonance spectroscopy, and increased copies of M2 mRNA as determined by Northern blot analysis. Two species of M2 mRNA were detected in rodent cell lines, a high-molecular-weight species of approximately 3.4 kb in hamster and rat cells and about 2.1 kb in mouse cells. The low molecular-weight M2 mRNA was about 1.6 kb in all rodent lines. Northern blot analysis showed that the mRNA for the other component of ribonucleotide reductase, M1, was not markedly elevated in the drug-resistant cells and existed as a single 3.1-kb species. Four of the five resistant lines contained an M2gene amplification as determined by Southern blot analysis, providing direct evidence to support earlier suggestions that hydroxyurea resistance is often accompanied by amplification of a ribonucleotide reductase gene. An increase in gene dosage was detected even in cells exhibiting only modest drug-resistance properties. No evidence for amplification of the M1gene of ribonucleotide reductase was found. In keeping with these observations with drug-resistant rodent lines, a human (HeLa) cell line resistant to hydroxyurea was also found to contain increased levels of two M2 mRNA species (about 3.4 and 1.6 kb) and exhibited M2gene amplification. One hamster cell line resembled the other resistant rodent lines in cellular characteristics but did not show amplification of either the M1or M2gene, providing an example of a drug-resistant mechanism in which an elevation of M2 mRNA has occurred without a concomitant increase in M2gene copy number.