Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Inhibitor Sml1 in Response to Iron Deficiency

Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox-active cofactor in many biological processes, including DNA replication and repair. Eukaryotic ribonucleotide reductases (RNRs) are Fe-dependent enzymes that catalyze deoxyribonucleoside diphosphate (dNDP) synthesis. We show here that the levels of the Sml1 protein, a yeast RNR large-subunit inhibitor, specifically decrease in response to both nutritional and genetic Fe deficiencies in a Dun1-dependent but Mec1/Rad53- and Aft1-independent manner. The decline of Sml1 protein levels upon Fe starvation depends on Dun1 forkhead-associated and kinase domains, the 26S proteasome, and the vacuolar proteolytic pathway. Depletion of core components of the mitochondrial iron-sulfur cluster assembly leads to a Dun1-dependent diminution of Sml1 protein levels. The physiological relevance of Sml1 downregulation by Dun1 under low-Fe conditions is highlighted by the synthetic growth defect observed between dun1Δ and fet3Δ fet4Δ mutants, which is rescued by SML1 deletion. Consistent with an increase in RNR function, Rnr1 protein levels are upregulated upon Fe deficiency. Finally, dun1Δ mutants display defects in deoxyribonucleoside triphosphate (dNTP) biosynthesis under low-Fe conditions. Taken together, these results reveal that the Dun1 checkpoint kinase promotes RNR function in response to Fe starvation by stimulating Sml1 protein degradation.     

The ribonucleotide reductase inhibitor,Sml1, is sequentially phosphorylated,ubiquitylated and degraded in response to DNA damage

Regulation of ribonucleotide reductase (RNR) is important for cell survival and genome integrity in the face of genotoxic stress. The Mec1/Rad53/Dun1 DNA damage response kinase cascade exhibits multifaceted controls over RNR activity including the regulation of the RNR inhibitor, Sml1. After DNA damage, Sml1 is degraded leading to the up-regulation of dNTP pools by RNR. Here, we probe the requirements for Sml1 degradation and identify several sites required for in vivo phosphorylation and degradation of Sml1 in response to DNA damage. Further, in a strain containing a mutation in Rnr1, rnr1-W688G, mutation of these sites in Sml1 causes lethality. Degradation of Sml1 is dependent on the 26S proteasome. We also show that degradation of phosphorylated Sml1 is dependent on the E2 ubiquitin-conjugating enzyme, Rad6, the E3 ubiquitin ligase, Ubr2, and the E2/E3-interacting protein, Mub1, which form a complex previously only implicated in the ubiquitylation of Rpn4.     

Identification of phosphorylation sites on the yeast ribonucleotide reductase inhibitor Sml1

Sml1 is a small protein in Saccharomyces cerevisiae which inhibits the activity of ribonucleotide reductase (RNR). RNR catalyzes the rate-limiting step of de novo dNTP synthesis. Sml1 is a downstream effector of the Mec1/Rad53 cell cycle checkpoint pathway. The phosphorylation by Dun1 kinase during S phase or in response to DNA damage leads to diminished levels of Sml1. Removal of Sml1 increases the population of active RNR, which raises cellular dNTP levels. In this study using mass spectrometry and site-directed mutagenesis, we have identified the region of Sml1 phosphorylation to be between residues 52 and 64 containing the sequence GSSASASASSLEM. This is the first identification of a phosphorylation sequence of a Dun1 biological substrate. This sequence is quite different from the consensus Dun1 phosphorylation sequence reported previously from peptide library studies. The specific phosphoserines were identified to be Ser(56), Ser(58), and Ser(60) by chemical modification of these residues to S-ethylcysteines followed by collision activated dissociation. To investigate further Sml1 phosphorylation, we constructed the single mutants S56A, S58A, S60A, and the triple mutant S56A/S58A/S60A and compared their degrees of phosphorylation with that of wild type Sml1. We observed a 90% decrease in the relative phosphorylation of S60A compared with that of wild type, a 25% decrease in S58A, and little or no decrease in the S56A mutant. There was no observed phosphate incorporation in the triple mutant, suggesting that Ser(56), Ser(58), and Ser(60) in Sml1 are the sites of phosphorylation. Further mutagenesis studies reveal that Dun1 kinase requires an acidic residue at the +3 position, and there is cooperativity between the phosphorylation sites. These results show that Dun1 has a unique phosphorylation motif.     

Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality

In budding yeast, MEC1 and RAD53 are essential for cell growth. Previously we reported that mec1 or rad53 lethality is suppressed by removal of Sml1, a protein that binds to the large subunit of ribonucleotide reductase (Rnr1) and inhibits RNR activity. To understand further the relationship between this suppression and the Sml1-Rnr1 interaction, we randomly mutagenized the SML1 open reading frame. Seven mutations were identified that did not affect protein expression levels but relieved mec1 and rad53 inviability. Interestingly, all seven mutations abolish the Sml1 interaction with Rnr1, suggesting that this interaction causes the lethality observed in mec1 and rad53 strains. The mutant residues all cluster within the 33 C-terminal amino acids of the 104-amino-acid-long Sml1 protein. Four of these residues reside within an alpha-helical structure that was revealed by nuclear magnetic resonance studies. Moreover, deletions encompassing the N-terminal half of Sml1 do not interfere with its RNR inhibitory activity. Finally, the seven sml1 mutations also disrupt the interaction with yeast Rnr3 and human R1, suggesting a conserved binding mechanism between Sml1 and the large subunit of RNR from different species.     

Inactivation of Ku-mediated end joining suppresses mec1Delta lethality by depleting the ribonucleotide reductase inhibitor Sml1 through a pathway controlled by Tel1 kinase and the Mre11 complex

RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and DNA damage checkpoint responses. Their lethality can be suppressed by increasing the intracellular pool of deoxynucleotide triphosphates. We report that deletion of YKU70 or YKU80 suppresses mec1Delta, but not rad53Delta, lethality. We show that suppression of mec1Delta lethality is not due to Ku--associated telomeric defects but rather results from the inability of Ku- cells to efficiently repair DNA double strand breaks by nonhomologous end joining. Consistent with these results, mec1Delta lethality is also suppressed by lif1Delta, which like yku70Delta and yku80Delta, prevents nonhomologous end joining. The viability of yku70Delta mec1Delta and yku80Delta mec1Delta cells depends on the ATM-related Tel1 kinase, the Mre11-Rad50-Xrs2 complex, and the DNA damage checkpoint protein Rad9. We further report that this Mec1-independent pathway converges with the Rad53/Dun1-regulated checkpoint kinase cascade and leads to the degradation of the ribonucleotide reductase inhibitor Sml1.     

Sml1p is a dimer in solution: characterization of denaturation and renaturation of recombinant Sml1p

Sml1p is a small 104-amino acid protein from Saccharomyces cerevisiae that binds to the large subunit (Rnr1p) of the ribonucleotide reductase complex (RNR) and inhibits its activity. During DNA damage, S phase, or both, RNR activity must be tightly regulated, since failure to control the cellular level of dNTP pools may lead to genetic abnormalities, such as genome rearrangements, or even cell death. Structural characterization of Sml1p is an important step in understanding the regulation of RNR. Until now the oligomeric state of Sml1p was unknown. Mass spectrometric analysis of wild-type Sml1p revealed an intermolecular disulfide bond involving the cysteine residue at position 14 of the primary sequence. To determine whether disulfide bonding is essential for Sml1p oligomerization, we mutated the Cys14 to serine. Sedimentation equilibrium measurements in the analytical ultracentrifuge show that both wild-type and C14S Sml1p exist as dimers in solution, indicating that the dimerization is not a result of a disulfide bond. Further studies of several truncated Sml1p mutants revealed that the N-terminal 8-20 residues are responsible for dimerization. Unfolding/refolding studies of wild-type and C14S Sml1p reveal that both proteins refold reversibly and have almost identical unfolding/refolding profiles. It appears that Sml1p is a two-domain protein where the N-terminus is responsible for dimerization and the C-terminus for binding and inhibiting Rnr1p activity.     

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.     

Cadmium inhibits the protein degradation of Sml1 by inhibiting the phosphorylation of Sml1 in Saccharomyces cerevisiae

Cadmium is a toxic metal, and the mechanism of cadmium toxicity in living organisms has been well studied. Here, we used Saccharomyces cerevisiae as a model system to examine the detailed molecular mechanism of cell growth defects caused by cadmium. Using a plate assay of a yeast deletion mutant collection, we found that deletion of SML1, which encodes an inhibitor of Rnr1, resulted in cadmium resistance. Sml1 protein levels increased when cells were treated with cadmium, even though the mRNA levels of SML1 remained unchanged. Using northern and western blot analyses, we found that cadmium inhibited Sml1 degradation by inhibiting Sml1 phosphorylation. Sml1 protein levels increased when cells were treated with cadmium due to disruption of the dependent protein degradation pathway. Furthermore, cadmium promoted cell cycle progression into the G2 phase. The same result was obtained using cells in which SML1 was overexpressed. Deletion of SML1 delayed cell cycle progression. These results are consistent with Sml1 accumulation and with growth defects caused by cadmium stress. Interestingly, although cadmium treatment led to increase Sml1 levels, intracellular dNTP levels also increased because of Rnr3 upregulation due to cadmium stress. Taken together, these results suggest that cadmium specifically affects the phosphorylation of Sml1 and that Sml1 accumulates in cells.     

Molecular Basis of the Essential S Phase Function of the Rad53 Checkpoint Kinase

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.     

Characterization of monomeric and dimeric forms of recombinant Sml1p-histag protein by electrospray mass spectrometry.

Sml1p is small protein that binds to and inhibits the activity of ribonucleotide reductase (RNR)3, a protein enzyme complex that controls the balance and level of the cellular deoxynucleotide diphosphate pools that are critical for DNA synthesis and repair. In this respect, Sml1p is a checkpoint protein whose function is to regulate the activity of the large subunit of RNR (Rnr1p). Sml1p is thought to be regulated by the MEC1/RAD53 cell cycle checkpoint pathway. Neither the structure of Sml1p nor its complex to Rnr1p is well known. In this report, we describe how a recombinant Sml1p-histag protein (in both monomeric and dimeric forms) can be characterized with electrospray mass spectrometry. Mass spectrometry can play a vital role in the study of the Sml1p-Rnr1p complex by: (1) confirming the identities and purities of recombinant proteins such as Sm1lp-histag (with mass accuracy and resolution far superior to SDS-PAGE) and (2) verifying the presence or absence of PTM, chemical modifications, or metal-ion binding to the protein species, which may alter the function and binding of the protein partners.     

Hug1 is an intrinsically disordered protein that inhibits ribonucleotide reductase activity by directly binding Rnr2 subunit

Rad53 is a conserved protein kinase with a central role in DNA damage response and nucleotide metabolism. We observed that the expression of a dominant-lethal form of RAD53 leads to significant expression changes for at least 16 genes, including the RNR3 and the HUG1 genes, both of which are involved in the control of nucleotide metabolism. We established by multiple biophysical and biochemical approaches that Hug1 is an intrinsically disordered protein that directly binds to the small RNR subunit Rnr2. We characterized the surface of interaction involved in Hug1 binding to Rnr2, and we thus defined a new binding region to Rnr2. Moreover, we show that Hug1 is deleterious to cell growth in the context of reduced RNR activity. This inhibitory effect of Hug1 on RNR activity depends on the binding of Hug1 to Rnr2. We propose a model in which Hug1 modulates Rnr2–Rnr1 association by binding Rnr2. We show that Hug1 accumulates under various physiological conditions of high RNR induction. Hence, both the regulation and the mode of action of Hug1 are different from those of the small protein inhibitors Dif1 and Sml1, and Hug1 can be considered as a regulator for fine-tuning of RNR activity.     

Dif1 is a DNA-damage-regulated facilitator of nuclear import for ribonucleotide reductase

The control of dNTP concentrations is critical to the fidelity of DNA synthesis and repair. One level of regulation is through subcellular localization of ribonucleotide reductase. In Saccharomyces cerevisiae, the small subunit Rnr2-Rnr4 is nuclear, whereas the large subunit Rnr1 is cytoplasmic. In response to S phase or DNA damage, Rnr2-Rnr4 enters the cytoplasm to bind Rnr1, forming an active complex. We previously reported that Wtm1 anchors Rnr2-Rnr4 in the nucleus. Here, we identify DIF1, which regulates localization of Rnr2-Rnr4. Dif1 binds directly to the Rnr2-Rnr4 complex through a conserved Hug domain to drive nuclear import. Dif1 is both cell-cycle and DNA-damage regulated, the latter of which occurs via the Mec1-Dun1 pathway. In response to DNA damage, Dun1 directly phosphorylates Dif1, which both inactivates and degrades Dif1 and allows Rnr2-Rnr4 to become cytoplasmic. We propose that Rnr2-Rnr4 nuclear localization is achieved by a dynamic combination of Wtm1-mediated nuclear retention to limit export and regulated nuclear import through Dif1.     

Regulation of ribonucleotide reductase in response to iron deficiency

Ribonucleotide reductase (RNR) is an essential enzyme required for DNA synthesis and repair. Although iron is necessary for class Ia RNR activity, little is known about the mechanisms that control RNR in response to iron deficiency. In this work, we demonstrate that yeast cells control RNR function during iron deficiency by redistributing the Rnr2-Rnr4 small subunit from the nucleus to the cytoplasm. Our data support a Mec1/Rad53-independent mechanism in which the iron-regulated Cth1/Cth2 mRNA-binding proteins specifically interact with the WTM1 mRNA in response to iron scarcity and promote its degradation. The resulting decrease in the nuclear-anchoring Wtm1 protein levels leads to the redistribution of the Rnr2-Rnr4 heterodimer to the cytoplasm, where it assembles as an active RNR complex and increases deoxyribonucleoside triphosphate levels. When iron is scarce, yeast selectively optimizes RNR function at the expense of other non-essential iron-dependent processes that are repressed, to allow DNA synthesis and repair.