Ribonucleotide reductase: regulation, regulation, regulation

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Abstract

Ribonucleotide reductase (RNR) catalyses the rate limiting step in the production of deoxyribonucleotides needed for DNA synthesis. It is composed of two dissimilar subunits, R1, the large subunit containing the allosteric regulatory sites, and R2, the small subunit containing a binuclear iron center and a tyrosyl free radical. Recent isolation of the mammalian and yeast RNR genes has shown that, in addition to the well documented allosteric regulation, the synthesis of the enzyme is also tightly regulated at the level of transcription. The mRNAs for both subunits are cell-cycle regulated and, in yeast, inducible by DNA damage. Yeast encode a second large subunit gene, RNR3, that is expressed only in the presence of DNA damage. This regulation is thought to provide a metabolic state that facilitates DNA replicational repair processes.

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The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae.

Brian T. Weinert, S Hartwell (1988)

Cell division is arrested in many organisms in response to DNA damage. Examinations of the genetic basis for this response in the yeast Saccharomyces cerevisiae indicate that the RAD9 gene product is essential for arrest of cell division induced by DNA damage. Wild-type haploid cells irradiated with x-rays either arrest or delay cell division in the G2 phase of the cell cycle. Irradiated G1 and M phase haploid cells arrest irreversibly in G2 and die, whereas irradiated G2 phase haploid cells delay in G2 for a time proportional to the extent of damage before resuming cell division. In contrast, irradiated rad9 cells in any phase of the cycle do not delay cell division in G2, but continue to divide for several generations and die. However, efficient DNA repair can occur in irradiated rad9 cells if irradiated cells are blocked for several hours in G2 by treatment with a microtubule poison. The RAD9-dependent response detects potentially lethal DNA damage and causes arrest of cells in G2 until such damage is repaired.

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Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase.

J. Davis, S Elledge (1990)

Ribonucleotide reductase activity is essential for progression through the cell cycle, catalyzing the rate-limiting step for the production of deoxyribonucleotides needed for DNA synthesis. The enzymatic activity of the enzyme fluctuates in the cell cycle with an activity maximum in S phase. We have identified and characterized two Saccharomyces cerevisiae genes encoding the regulatory subunit of ribonucleotide reductase, RNR1 and RNR3. They share approximately 80% amino acid identity with each other and 60% with the mammalian homolog, M1. Genetic disruption reveals that the RNR1 gene is essential for mitotic viability, whereas the RNR3 gene is not essential. A high-copy-number clone of RNR3 is able to suppress the lethality of rnr1 mutations. Analysis of mRNA levels in cell-cycle-synchronized cultures reveals that the RNR1 mRNA is tightly cell-cycle regulated, fluctuating 15- to 30-fold, and is coordinately regulated with the POL1 mRNA, being expressed in the late G1 and S phases of the cell cycle. Progression from the alpha-factor-induced G1 block to induction of RNR1 mRNA is blocked by cycloheximide, further defining the requirement for protein synthesis in the G1- to S-phase transition. Both RNR1 and RNR3 transcripts are inducible by treatments that damage DNA, such as 4-nitroquinoline-1-oxide and methylmethanesulfonate, or block DNA replication, such as hydroxyurea. RNR1 is inducible 3- to 5-fold, and RNR3 is inducible greater than 100-fold. When MATa cells are arrested in G1 by alpha-factor, RNR1 and RNR3 mRNA is still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. Inhibition of ribonucleotide reductase activity by hydroxyurea treatment results in arrest of the cell cycle in S phase as large budded, uninucleate cells. This specific cell-cycle arrest is independent of the RAD9 gene, defining a separate pathway for the coordination of DNA synthesis and cell-cycle progression.

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Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements.

K Nasmyth, David Shore (1987)

A DNA binding protein (RAP1, previously called SBF-E) has been shown to bind to putative regulatory sites at both yeast mating-type silencers, yet is not the product of genetically identified regulators of the silent loci. Here, we report the purification of RAP1 by DNA affinity chromatography, and the isolation of its gene from a lambda gt11 genomic library using antibodies raised against the protein. Disruption of the chromosomal copy of this gene is lethal. We show that RAP1 protein also binds in vitro to the upstream activation site (UAS) of MAT alpha and ribosomal protein genes. In addition, we show that two different UAS-associated RAP1 binding sites can substitute in vivo for a silencer binding site. Our results suggest that RAP1 may be a transcriptional regulator that can play a role in either repression or activation of transcription, depending upon the context of its binding site.