Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1

Abstract

Ribonucleotide reductase maintains cellular deoxyribonucleotide pools and is thus tightly regulated during the cell cycle to ensure high fidelity in DNA replication. The Sml1 protein inhibits ribonucleotide reductase activity by binding to the R1 subunit. At the completion of each turnover cycle, the active site of R1 becomes oxidized and subsequently regenerated by a cysteine pair (CX 2C) at its C-terminal domain (R1-CTD). Here we show that R1-CTD acts in trans to reduce the active site of its neighboring monomer. Both Sml1 and R1-CTD interact with the N-terminal domain of R1 (R1-NTD), which involves a conserved two-residue sequence motif in the R1-NTD. Mutations at these two positions enhancing the Sml1-R1 interaction cause SML1-dependent lethality. These results point to a model whereby Sml1 competes with R1-CTD for association with R1-NTD to hinder the accessibility of the CX 2C motif to the active site for R1 regeneration.deoxyribonucleotides ͉ DNA replication T he maintenance of adequate and balanced dNTP levels is critical for faithful DNA replication and repair and for the survival of all organisms (1-5). A major target of dNTP pool regulation is ribonucleotide reductase (RNR) that catalyzes the essential step of converting ribonucleoside diphosphates to the corresponding deoxy forms in dNTP biosynthesis (6). Class I RNRs are conserved from eubacteria to eukaryotes and comprise two subunits: R1 and R2. R1 (␣ 2 in Escherichia coli and ␣ 2 , ␣ 4 , and ␣ 6 in eukaryotes; ref. 7) contains the active site as well as binding sites for allosteric effectors; R2 (␤ 2 ) houses a diferric-tyrosyl radical required for catalysis (8). These subunits can be regulated by allostery (1), transcription (9), subcellular compartmentalization (10 -13), and protein inhibitor interaction (14, 15).The 104-residue Saccharomyces cerevisiae Sml1 protein was originally identified as an RNR inhibitor based on the finding that loss of SML1 function suppresses the lethality of cells lacking the checkpoint kinases Mec1 or Rad53 by increasing cellular dNTP levels (15). Sml1 is phosphorylated and degraded during S phase and after DNA damage in a checkpointdependent manner to relieve RNR inhibition (16). The inhibition of R1 by Sml1 depends on Sml1-R1 association because mutations in SML1 disrupting its R1-binding ability abolish the inhibition (17). Crystallographic studies of the R1s from E. coli and S. cerevisiae reveal three domains in the protein: the N-terminal helical domain, the 10-stranded ␣/␤-barrel domain, and the C-terminal domain of less-defined structure (18,19). The active site is located in the center of the protein between the N-terminal and the barrel domains, wherein a redox-active cysteine pair (Cys-225/Cys-462 of the E. coli R1 and Cys-218/ Cys-443 of the yeast R1) converts from a free dithiol form in the reduced R1 (active form) to a disulfide-bonded form in the oxidized R1 (inactive form) after each reduction cycle (20). This disulfide bond is reduced to regenerate an active R1 for the subsequent catalytic cycles (21...

Nonself Recognition Through Intermolecular Disulfide Bond Formation of Ribonucleotide Reductase in Neurospora

“…We propose that an aberrant disulfide bond between cysteines in the UN-24 PA C terminus (C907 and C918/C921) and the UN-24 OR catalytic site (e.g., C444) leads to an aberrant heterodimeric complex, which in turn may lead to a toxic higher order complex. This is consistent with evidence from yeast that the C-terminal domain of one R1 subunit acts in trans to reduce the active site of the adjoining R1 subunit (Zhang et al 2007). These intermolecular disulfide bonds could align the PA and OR UN-24 proteins and facilitate new covalent or noncovalent intermolecular interactions that may lead to a complex or aggregate that is resistant to the denaturants used in this study (i.e., DTT), which have been used previously to reduce disulfide bonds (Scigelova et al 2001).…”

“…Removal of six residues from the C terminus end [hygunPA (788-917)], including the putative redox-active and catalytically required C terminus cysteine pair, resulted in a loss of incompatibility activity. These residues lie in the putative flexible C terminus arm of R1 as defined in yeast (Xu et al 2006;Zhang et al 2007).…”

“…Subsequently, the active site disulfide bond is transferred to C754 and C759 of the C terminus region, which is then reduced by glutaredoxin or thioredoxin to regenerate the enzyme (Aberg et al 1989;Mao et al 1992). Although the mechanism by which this disulfide bond transfer occurs is unknown, it has been demonstrated that, in yeast, the labile structure of the C terminus allows for the C-terminal cysteine residues of one R1 subunit to act in trans to reduce the active site of the neighboring R1 subunit (Zhang et al 2007).…”

Essential role of Tip60-dependent recruitment of ribonucleotide reductase at DNA damage sites in DNA repair during G1 phase

“…We first generated RRM1 mutants that lack the ability to bind Tip60 but retain RNR activity. Given that the C-terminal CXXC motif of RRM1 is important for RNR function (Zhang et al 2007), we constructed RRM1 mutants containing the CXXC motif but lacking Tip60-binding ability (D761-786 and A776CD781-C) (Fig. 2D).…”

Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

“…In S. cerevisiae, the Sml1 inhibitor binds to the R1 subunit (Zhao et al 2000;Zhang et al 2007) and prevents dNTP synthesis by inserting its C-terminal aromatic residue into a cleft usually occupied by the C-terminal residue of R2. Each S phase, Mec1 kinase activates the downstream Dun1 kinase to promote Sml1 degradation (Zhao and Rothstein 2002), likely by phosphorylating serine residues within the Sml1 phospho-degron (SML box) (see Fig.…”