Molecular characterization of tobacco ribonucleotide reductase RNR1 and RNR2 cDNAs and cell cycle-regulated expression in synchronized plant cells

Eukaryotic ribonucleotide reductase (RNR), the enzyme involved in the synth esis of the deoxyribonucleotides, consists of two R1 and R2 subunits whose activities and gene expression are differentially regulated during the cell cycle and are preferentially induced at the G1/S transition. We have isola ted three cDNA clones from a tobacco S phase library, two encoding the larg e RI subunit, the first cloned in plants, and one encoding the small R2 sub unit. From Southern blot hybridization we deduce that RNR2 is encoded by a single-copy gene whereas RNR1 is encoded by a small multigene family. The l evel of RNR mRNA is cell-cycle regulated showing a maximum in S phase. In m id-S phase, RNR2 transcripts show a higher maximum level than RNR1 transcri pts. Analysis of the effects of various cell cycle inhibitors added to fres hly subcultured stationary phase cells leads to the conclusion that RNR gen e induction at the entry of the cells into the cell cycle takes place in la te G1-early S phase. Addition of DNA synthesis-blocking agents to cycling c ells synchronized in mid-S phase resulted in an enhancement of RNR transcri pt level, thus suggesting that RNR gene expression may be linked to the DNA synthesis rate by a feedback-like regulatory mechanism.