Ribonucleotide reductase catalyzes a crucial step in de novo DNA synthesis and is allosterically controlled by relative levels of dNTPs to maintain a balanced pool of deoxynucleoside triphosphates in the cell. In eukaryotes, the enzyme comprises a heterooligomer of ␣2 and ␤2 subunits. The ␣ subunit, Rnr1, contains catalytic and regulatory sites. Here, we report the only x-ray structures of the eukaryotic ␣ subunit of ribonucleotide reductase from Saccharomyces cerevisiae. The structures of the apo-, AMPPNP only-, AMPPNP-CDP-, AMPPNP-UDP-, dGTP-ADP-and TTP-GDP-bound complexes give insight into substrate and effector binding and specificity cross-talk. These are Class I structures with the only fully ordered catalytic sites, including loop 2, a stretch of polypeptide that spans specificity and catalytic sites, conferring specificity. Binding of specificity effector rearranges loop 2; in our structures, this rearrangement moves P294, a residue unique to eukaryotes, out of the catalytic site, accommodating substrate binding. Substrate binding further rearranges loop 2. Cross-talk, by which effector binding regulates substrate preference, occurs largely through R293 and Q288 of loop 2, which are analogous to residues in Thermotoga maritima that mediate cross-talk. However loop-2 conformations and residue-substrate interactions differ substantially between yeast and T. maritima. In most effector-substrate complexes, water molecules help mediate substrate-loop 2 interactions. Finally, the substrate ribose binds with its 3 hydroxyl closer than its 2 hydroxyl to C218 of the catalytic redox pair. We also see a conserved water molecule at the catalytic site in all our structures, near the ribose 2 hydroxyl.ribonucleotide reductase ͉ allosteric regulation ͉ crystallography ͉ DNA synthesis ͉ dNTP pools E ukaryotic ribonucleotide reductase (RNR) is an enzyme composed of ␣ 2 and ␤ 2 subunits that catalyzes a crucial step of de novo DNA synthesis by converting nucleoside diphosphates to deoxynucleoside diphosphates (1, 2). Tight control of dNTP pools is vital for cell viability; because of the crucial role RNR plays in balancing the relative levels of dNTPs, it is highly regulated transcriptionally (3), allosterically (4-6), by compartmentalization of the various subunits within the cell (7,8), and, in Saccharomyces cerevisiae, by its protein inhibitor Sml1 (9-12). The molecular basis for these processes is not fully understood.Rnr1, the ␣ subunit of RNR, contains the catalytic site, the substrate-specificity site, and the activity site (Fig. 1A), whereas the ␤ subunit houses a tyrosyl radical required for RNR activity (13,14). An elegant mechanism of specificity cross-talk determines substrate preference based on the nucleotide effector bound at the specificity site (4, 15, 16). Brown and Reichard (2) have proposed that the effectors ATP and dATP bind at the activity site and activate or inhibit, respectively. They also bind at the specificity site and select for pyrimidine substrates, whereas thymin triphosphate (TTP) and dGT...

Human testis determination is initiated by SRY (sex determining region on Y chromosome). Mutations in SRY cause gonadal dysgenesis with female somatic phenotype. Two subtle variants (V60L and I90M in the high-mobility group box) define inherited alleles shared by an XY sterile daughter and fertile father. Whereas specific DNA binding and bending are unaffected in a rat embryonic pre-Sertoli cell line, the variants exhibited selective defects in nucleocytoplasmic shuttling due to impaired nuclear import (V60L; mediated by Exportin-4) or export (I90M; mediated by chromosome region maintenance 1). Decreased shuttling limits nuclear accumulation of phosphorylated (activated) SRY, in turn reducing occupancy of DNA sites regulating Sertoli-cell differentiation [the testis-specific SRY-box 9 (Sox9) enhancer]. Despite distinct patterns of biochemical and cell-biological perturbations, V60L and I90M each attenuated Sox9 expression in transient transfection assays by twofold. Such attenuation was also observed in studies of V60A, a clinical variant associated with ovotestes and hence ambiguity between divergent cell fates. This shared twofold threshold is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9. Our results demonstrate that nucleocytoplasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and importdependent phosphorylation are compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects in the nucleocytoplasmic shuttling of SRY suggests that its transcriptional activity lies near the edge of developmental ambiguity.organogenesis | sex determination | gonadogenesis | gene-regulatory network | protein-DNA interaction

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Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. Crucial for rapidly dividing cells, RNR is a target for cancer therapy. In eukaryotes, RNR comprises a heterooligomer of ␣2 and ␤2 subunits. Rnr1, the ␣ subunit, contains regulatory and catalytic sites; Rnr2, the ␤ subunit (in yeast, a heterodimer of Rnr2 and Rnr4), houses the diferric-tyrosyl radical crucial for catalysis. Here, we present three x-ray structures of eukaryotic Rnr1 from Saccharomyces cerevisiae: one bound to gemcitabine diphosphate (GemdP), the active metabolite of the mechanism-based chemotherapeutic agent gemcitabine; one with an Rnr2-derived peptide, and one with an Rnr4-derived peptide. Our structures reveal that GemdP binds differently from its analogue, cytidine diphosphate; because of unusual interactions of the geminal fluorines, the ribose and base of GemdP shift substantially, and loop 2, which mediates substrate specificity, adopts different conformations when binding to GemdP and cytidine diphosphate. The Rnr2 and Rnr4 peptides, which block RNR assembly, bind differently from each other but have unique modes of binding not seen in prokaryotic RNR. The Rnr2 peptide adopts a conformation similar to that previously reported from an NMR study for a mouse Rnr2-based peptide. In yeast, the Rnr2 peptide binds at subsites consisting of residues that are highly conserved among yeast, mouse, and human Rnr1s, suggesting that the mode of Rnr1-Rnr2 binding is conserved among eukaryotes. These structures provide new insights into subunit assembly and a framework for structure-based drug design targeting RNR.allosteric regulation ͉ crystallography ͉ dNTP ͉ chemotherapy ͉ gemcitabine R ibonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, essential precursors of DNA synthesis. Crucial for rapidly proliferating cells, RNR is a target for anticancer (1, 2) and antiviral (2, 3) drugs. Gemcitabine, an analogue of deoxycytidine (2Ј-2Ј-difluorodeoxycytidine), is sequentially phosphorylated to the 5Ј-monophosphate form by deoxycytidine kinase and to difluorodeoxycytidine 5Ј-diphosphate (GemdP) by uridylate-cytidylate monophosphate kinase. In the presence of reductants, GemdP inactivates Rnr1. In the absence of reductants, with prereduced Rnr1 and Rnr2, inhibition occurs from the loss of the tyrosyl radical in Rnr2 (1). Recently, GemdP has been shown to inactivate both human R1 and R2 (JoAnne Stubbe, personal communication). Inhibition of RNR by GemdP leads to reduction of the pool of deoxyribonucleotide 5Ј-diphosphates available for DNA synthesis, presumably favoring incorporation of the gemcitabine triphosphate metabolite by DNA polymerase ␣, preventing chain elongation (4, 5).RNRs require unusual metallocofactors to initiate radicalbased nucleotide reduction and are divided into three classes based on their cofactor. Class I RNR, found in all eukaryotes, is a heterooligomer of ␣ 2 and ␤ 2 subunits (6). In eukaryotes, the ␣ subunit, called Rnr1, c...

The male program of therian mammals is determined by Sry, a transcription factor encoded by the Y chromosome. Specific DNA binding is mediated by a high mobility group (HMG) box. Expression of Sry in the gonadal ridge activates a Sox9-dependent gene regulatory network leading to testis formation. A subset of Sry alleles in superfamily Muroidea (order Rodentia) is remarkable for insertion of an unstable DNA microsatellite, most commonly encoding (as in mice) a CAG repeat-associated glutamine-rich domain. We provide evidence, based on an embryonic pre-Sertoli cell line, that this domain functions at a threshold length as a genetic capacitor to facilitate accumulation of variation elsewhere in the protein, including the HMG box. The glutamine-rich domain compensates for otherwise deleterious substitutions in the box and absence of nonbox phosphorylation sites to ensure occupancy of DNA target sites. Such compensation enables activation of a male transcriptional program despite perturbations to the box. Whereas human SRY requires nucleocytoplasmic shuttling and coupled phosphorylation, mouse Sry contains a defective nuclear export signal analogous to a variant human SRY associated with inherited sex reversal. We propose that the rodent glutamine-rich domain has (i) fostered accumulation of cryptic intragenic variation and (ii) enabled unmasking of such variation due to DNA replicative slippage. This model highlights genomic contingency as a source of protein novelty at the edge of developmental ambiguity and may underlie emergence of nonSry-dependent sex determination in the radiation of Muroidea.nucleocytoplasmic trafficking | protein-DNA recognition | sexual dimorphism | transcriptional activation | triplet expansion

Male sex determination in mammals is initiated by SRY, a Y-encoded transcription factor. The protein contains a high-mobility-group (HMG) box mediating sequence-specific DNA bending. Mutations causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box and ordinarily arise de novo. Rare inherited variants lead to male development in one genetic background (the father) but not another (his sterile XY daughter). De novo and inherited mutations occur at an invariant Tyr adjoining the motif’s basic tail (box position 72; Y127 in SRY). In SRY-responsive cell lines CH34 and LNCaP, de novo mutations Y127H and Y127C reduced SRY activity (as assessed by transcriptional activation of principal target gene Sox9) by 5- and 8-fold, respectively. Whereas Y127H impaired testis-specific enhancer assembly, Y127C caused accelerated proteasomal proteolysis; activity was in part rescued by proteasome inhibition. Inherited variant Y127F was better tolerated: its expression was unperturbed, and activity was reduced by only twofold, a threshold similar to other inherited variants. Biochemical studies of wild-type (WT) and variant HMG boxes demonstrated similar specific DNA affinities (within a twofold range), with only subtle differences in sharp DNA bending as probed by permutation gel electrophoresis and fluorescence resonance-energy transfer (FRET); thermodynamic stabilities of the free boxes were essentially identical. Such modest perturbations are within the range of species variation. Whereas our cell-based findings rationalize the de novo genotype-phenotype relationships, a molecular understanding of inherited mutation Y127F remains elusive. Our companion study uncovers cryptic biophysical perturbations suggesting that the para-OH group of Y127 anchors a novel water-mediated DNA clamp.

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.

Background: Testis-determining factor SRY provides a model of bent protein⅐DNA complex. Results: Mutation of an invariant Trp perturbs multiple biochemical, cellular, and transcriptional activities. Conclusion: Folding and function of a sequence-specific HMG box require a core "aromatic buttress." Significance: Mutations in SRY causing human sex reversal probe the architecture and evolution of a DNA-bending motif.