Controlled Protein Degradation Regulates Ribonucleotide Reductase Activity in Proliferating Mammalian Cells during the Normal Cell Cycle and in Response to DNA Damage and Replication Blocks

Andrei Chabes,Lars Thelander

doi:10.1074/jbc.m000799200

Abstract

Ribonucleotide reductase (RNR) plays a central role in the formation and control of the optimal levels of deoxyribonucleoside triphosphates, which are required for DNA replication and DNA repair processes. Mammalian RNRs are composed of two nonidentical subunits, proteins R1 and R2. The levels of the limiting R2 protein control overall RNR activity during the mammalian cell cycle, being undetectable in G(1) phase and increasing in S phase. We show that in proliferating mammalian cells, the transcription of the R2 gene, once activated in the beginning of S phase, reaches its maximum 6-7 h later and then declines. Surprisingly, DNA damage and replication blocks neither increase nor prolong the R2 promoter activity in S phase. Instead, the cell cycle activity of the mammalian enzyme is controlled by an S phase/DNA damage-specific stabilization of the R2 protein, which is effective until cells pass into mitosis.

Highlights

Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from the corresponding ribonucleotides, which is the rate-limiting step in the production of the precursors for DNA synthesis [1]
Effects of DNA Damage and Replication Blocks on the Activity of the Mouse R2 Gene Promoter in Proliferating Cells—The activity of the mouse R2 promoter during S phase was assayed using an R2 promoter-luciferase reporter gene construct, which was stably transformed into mouse Balb/3T3 fibroblasts
To test whether the R2 promoter can be activated at the time of maximal DNA synthesis, we applied hydroxyurea, aphidicolin, or UV light to cells synchronously passing through the S phase 18 h after the release from serum starvation (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Reagents and Proteins—[␥-32P]ATP and [35S]methionine/cysteine (PRO-MIX [35S]cell labeling mix) were obtained from Amersham Pharmacia Biotech; hydroxyurea and roscovitine were from Calbiochem; and aphidicolin, nocodazole, and LLnL were from Sigma. Mouse recombinant R1 and R2 proteins were prepared from BL21(DE3)pLysS bacteria containing the plasmids pETM1 or pETM2 and purified to homogeneity according to Refs. Vector Construction—The R2 promoter-luciferase reporter gene construct was created by ligating a 1517-base pair PvuII-PvuII R2 promoter fragment (nucleotides Ϫ1500 to ϩ17 relative to the major transcription start) into the pGL3 vector (Promega) digested with EcoICRI and NcoI endonucleases and blunted with the Klenow fragment. The pM2Hind plasmid containing the entire mouse R2 gene was partly digested with ClaI (at nucleotide Ϫ1006) and NdeI. The created fragments were ligated into the native R2 gene instead of the original fragment, which resulted in either the replacement of the Ser residue by Ala (S20A mutant) or the deletion of the residues 2–20

Regulation of Mammalian Ribonucleotide Reductase

RESULTS

DISCUSSION