Cell Cycle-dependent Expression of Mammalian Ribonucleotide

Consistent with its specialized role in DNA synthesis, the activity of ribonucleotide reductase is cell cycledependent, reaching its maximum during S-phase. This paper demonstrates, however, that levels of the two protein subunits, M1 and M2, of this enzyme vary independently of one another. The level of protein M1 was determined by use of a two-site monoclonal antibody-enzyme immunoassay and found to be constant throughout the cell cycle in bovine kidney MDBK cells. Pulse-chase experiments showed that the half-life of protein M1 was 15 h. This contrasts with our previous results demonstrating an S-phase-correlated increase in the concentration of protein M2 and a half-life of this subunit of 3 h. Therefore, ribonucleotide reductase is controlled during the cell cycle by the level of protein M2. Ribonucleotide reductase catalyzes the first unique step on the biochemical pathway leading to DNA synthesis (I, 2). In mammalian cells, the enzyme consists of two nonidentical subunits, proteins M1 and M2. Protein M1 is a dimer of M, 170,000, which contains nucleotide binding sites and is responsible for the complex allosteric regulation of the enzyme (3). Protein M2, a dimer of M, 88,000, contains stoichiometric amounts of non-heme iron and a unique tyrosyl free radical, essential for activity (4-6). To study the regulation of ribonucleotide reduction during the cell cycle, a method was developed to measure directly the levels of protein M2 in whole cells, using EPR spectroscopy to quantify the M2-specific tyrosyl free radical. A 3-7-fold increase in the concentration of active protein M2 was observed when cells passed from the GI- to the S-phase of the cell cycle (7). Since protein M1 has no unique physical quality, such as the tyrosyl radical of protein M2, its direct determination in intact cell has not been achieved. Estimations of protein M1 activity in crude extracts are dependent on the properties of the M2 “helper” protein, and the assay is easily disturbed by the presence of nucleotides and nucleotide me