Yeast DNA damage-inducible Rnr3 has a very low catalytic activity strongly stimulated after the formation of a cross-talking Rnr1/Rnr3 complex.
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 cross-talk between the Rnr1 and Rnr3 polypeptides of the large subunit.
Domkin
Vladimir
V
Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
Thelander
Lars
L
Chabes
Andrei
A
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001483:Base Sequence; D001704:Biopolymers; D002384:Catalysis; D004249:DNA Damage; D017931:DNA Primers; D004271:DNA, Fungal / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D001704:Biopolymers; D017931:DNA Primers; D004271:DNA, Fungal; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases