Yeast DNA Damage-inducible Rnr3 Has a Very Low Catalytic Activity Strongly Stimulated after the Formation of a Cross-talking Rnr1/Rnr3 Complex

Abstract

The ribonucleotide reductase system in Saccharomyces cerevisiae includes four genes (RNR1 and RNR3 encoding the large subunit and RNR2 and RNR4 encoding the small subunit). RNR3 expression, nearly undetectable during normal growth, is strongly induced by DNA damage. Yet an rnr3 null mutant has no obvious phenotype even under DNA damaging conditions, and the contribution of RNR3 to ribonucleotide reduction is not clear. To investigate the role of RNR3 we expressed and characterized the Rnr3 protein. The in vitro activity of Rnr3 was less than 1% of the Rnr1 activity. However, a strong synergism between Rnr3 and Rnr1 was observed, most clearly demonstrated in experiments with the catalytically inactive Rnr1-C428A mutant, which increased the endogenous activity of Rnr3 by at least 10-fold. In vivo, the levels of Rnr3 after DNA damage never reached more than one-tenth of the Rnr1 levels. We propose that heterodimerization of Rnr3 with Rnr1 facilitates the recruitment of Rnr3 to the ribonucleotide reductase holoenzyme, which may be important when Rnr1 is limiting for dNTP production. In complex with inactive Rnr1-C428A, the activity of Rnr3 is controlled by effector binding to Rnr1-C428A. This result indicates crosstalk between the Rnr1 and Rnr3 polypeptides of the large subunit.DNA damage in eukaryotic cells leads to arrest of the cell cycle and activation of the genes involved in DNA repair (1). The DNA damage checkpoint pathway responsible for activation of DNA damage-inducible genes in yeast Saccharomyces cerevisiae has been the focus of intensive research during the past decade (2). The emerging conservation between the DNA damage checkpoint pathway in yeast and humans promotes a better understanding of this vital process (3, 4). One of the frequently used tools in the dissection of the DNA damage checkpoint pathway in S. cerevisiae has been the gene RNR3 (5). This gene is induced 5-10-fold (6 -8), and in some reports more than 100-fold (5), in response to DNA damage. In addition, it is not essential, and the rnr3 null mutation has no phenotype under all studied conditions (5). These properties of the RNR3 have been exploited in the identification of a number of important genes involved in the DNA damage checkpoint function such as CRT1, TUP1, SSN6, and DUN1 (9 -12). On the basis of homology with the RNR1 gene encoding the large subunit of yeast ribonucleotide reductase (RNR), 1 it has been proposed that RNR3 encodes a second large subunit of the yeast RNR (5). In support of this hypothesis, it was demonstrated that overexpression of RNR3 could rescue rnr1 null mutants (5).Ribonucleotide reductase is the rate-limiting enzyme in DNA precursor biosynthesis present in all living cells (13). All RNRs use free radical chemistry to reduce ribonucleotides to deoxyribonucleotides. Based on the nature of the cofactor providing the free radical for the reaction, RNRs were divided into three classes (14). Nearly all eukaryotes have an RNR belonging to class I in which the enzyme is thought to be an ␣ 2 ␤ 2 heterotetramer. ...

Single-Cell Analysis of Ribonucleotide Reductase Transcriptional and Translational Response to DNA Damage

“…This indicates that RNR1 mRNA numbers drop precipitously as cells initiate DNA synthesis. Also consistent with bulk studies (15,16,30), RNR3 mRNA was entirely absent throughout the cell cycle in undamaged log-phase cells and the cell cycle-dependent differences in RNR2 and RNR4 transcript numbers were relatively small, though significant, for RNR4 (Fig. 2B).…”

“…Each RNR gene was targeted by 40 20-nucleotide-long DNA oligonucleotide probes, each with a 3= Alexa Fluor 568 fluorophore. When designing probes, we used bioinformatics to eliminate any probe that can potentially cross-hybridize between genes like RNR1 and RNR3, which show large similarities (30) in nucleotide sequence (see Fig. S1 in the supplemental material).…”

The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis

“…This does not appear to be a general protein effect, since addition of truncated M45 protein or BSA resulted in no stimulation (data not shown). We tested the hypothesis that, in analogy with yeast Rnr3p (19), heterodimerization of R1 and M45 facilitates the recruitment of M45 to the holoenzyme. Therefore, we observed RNR activity when M45 was added to an assay system consisting of catalytically inactive mouse R1 C429A protein together with an excess of R2.…”

Autophagy-Dependent Regulation of the DNA Damage Response Protein Ribonucleotide Reductase 1

“…The degradation of excess Rnr1 protein during DNA damage may be required to promote the formation of an Rnr1-Rnr3 assembly in the final RNR complex, as opposed to an Rnr1-Rnr1 assembly. The Rnr1-Rnr3 assembly is reported to be the most catalytically active form (52). Ghaemmaghami and colleagues have reported that Rnr1 protein levels are approximately 200-fold higher than Rnr3 levels (53).…”