The fidelity of DNA replication and repair processes is critical for maintenance of genomic stability. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP production and thus plays an essential role in DNA synthesis. The level and activity of RNR are highly regulated by the cell cycle and DNA damage checkpoints, which maintain optimal dNTP pools required for genetic fidelity. RNRs are composed of a large subunit that binds the nucleoside diphosphate substrates and allosteric effectors and a small subunit that houses the di-iron tyrosyl radical cofactor essential for the reduction process. In Saccharomyces cerevisiae, there are two large subunits (Rnr1 and Rnr3) and two small subunits (Rnr2 and Rnr4). Here we report the subcellular localization of Rnr1-4 during normal cell growth and the redistribution of Rnr2 and Rnr4 in response to DNA damage and replicational stress. During the normal cell cycle, Rnr1 and Rnr3 are predominantly localized to the cytoplasm and Rnr2 and Rnr4 are predominantly present in the nucleus. Under genotoxic stress, Rnr2 and Rnr4 become redistributed to the cytoplasm in a checkpoint-dependent manner. Subcellular redistribution of Rnr2 and Rnr4 can occur in the absence of the transcriptional induction of the RNR genes after DNA damage and likely represents a posttranslational event. These results suggest a mechanism by which DNA damage checkpoint modulates RNR activity through the temporal and spatial regulation of its subunits. E ukaryotic cells have evolved complex surveillance mechanisms (i.e., checkpoints) to respond to genotoxic stress by arresting the cell cycle and inducing the transcription of genes that facilitate repair (1, 2). Failure of DNA damage response can result in genomic instability and cancer predisposition (3, 4). In mammalian cells the protein kinases ATM, ATR, and CHK2 are crucial for activating signaling pathways for cell survival after DNA damage (5-7). In the yeast Saccharomyces cerevisiae, the ATR homologue Mec1 and CHK2 homologue Rad53 are key regulators of cellular response to DNA damage, controlling the G 1 , S, and G 2 cell cycle checkpoints as well as transcriptional induction (8). Dun1, a protein kinase similar to Rad53, is also involved in these processes (9, 10). Among the best-studied transcriptional targets of the Mec1͞ Rad53͞Dun1 checkpoint pathway are the genes encoding ribonucleotide reductase (RNR; refs. 9 and 11-13).The enzymatic activity of RNR depends on the formation of a complex between two different subunits, R1 and R2. The large subunit R1 is a dimer and contains the active site for reduction of nucleoside diphosphate (NDP) substrates and the effector sites that control substrate specificity and enzymatic activity. The small subunit R2 is also a dimer that houses the di-iron tyrosyl radical (Y⅐) cofactor essential for NDP reduction. The active form of RNR is proposed to be a 1:1 complex of R1 and R2 (14-16).In budding yeast there are four RNR genes, two that code for a large subunit (RNR1 and RNR3) and two that code for a s...

The moenomycins are phosphoglycolipid antibiotics produced by Streptomyces ghanaensis and related organisms. The phosphoglycolipids are the only known active site inhibitors of the peptidoglycan glycosyltransferases, an important family of enzymes involved in the biosynthesis of the bacterial cell wall. Although these natural products have exceptionally potent antibiotic activity, pharmacokinetic limitations have precluded their clinical use. We previously identified the moenomycin biosynthetic gene cluster in order to facilitate biosynthetic approaches to new derivatives. Here we report a comprehensive set of genetic and enzymatic experiments that establish functions for the seventeen moenomycin biosynthetic genes involved in the synthesis moenomycin and variants. These studies reveal the order of assembly of the full molecular scaffold and define a subset of seven genes involved in the synthesis of bioactive analogs. This work will enable both in vitro and fermentation-based reconstitution of phosphoglycolipid scaffolds so that chemoenzymatic approaches to novel analogs can be explored.

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). This RNR is composed of two homodimeric subunits: R1 and R2. R1 binds the NDPs in the active site, and R2 harbors the essential di-iron tyrosyl radical (Y*) cofactor. In this paper, we used PELDOR, a method that detects weak electron-electron dipolar coupling, to make the first direct measurement of the distance between the two Y*'s on each monomer of R2. In the crystal structure of R2, the Y*'s are reduced to tyrosines, and consequently R2 is inactive. In R2, where the Y*'s assume a well-defined geometry with respect to the protein backbone, the PELDOR method allows measurement of a distance of 33.1 +/- 0.2 A that compares favorably to the distance (32.4 A) between the center of mass of the spin density distribution of each Y* on each R2 monomer from the structure. The experiments provide the first direct experimental evidence for two Y*'s in a single R2 in solution.

Peptidoglycan glycosyltransferases (PGTs) are highly conserved enzymes that catalyze the polymerization of Lipid II to form the glycan strands of bacterial murein. Because they play a key role in bacterial cell wall synthesis, these enzymes are potentially important antibiotic targets; however, their mechanisms are not yet understood. One longstanding question about these enzymes is whether they elongate glycan chains by adding subunits to the anomeric (reducing) end or to the 4-hydroxyl (non-reducing) end. We have developed an approach to test the direction of chain elongation that involves the use of nascent peptidoglycan chains which are blocked at their non-reducing ends. In the presence of the PGT domains of Staphylococcus aureus PBP2, Aquifex aeolicus PBP1A, Escherichia coli PBP1A or Escherichia coli PBP1B, these blocked substrates react with Lipid II to form longer glycan chains. These results establish that PGTs elongate nascent peptidoglycan chains by the addition of disaccharide subunits to the anomeric (reducing) end of the growing polymer.Peptidoglycan glycosyltransferases (PGTs) are highly conserved bacterial enzymes that catalyze the polymerization of a disaccharide called Lipid II (Figure 1) to form the glycan strands of peptidoglycan. PGTs are regarded as desirable antibiotic targets because they are extracellular, they do not have eukaryotic counterparts, and they play an essential role in a validated therapeutic pathway. 1 Although their importance has been appreciated for decades, the mechanism of glycan chain polymerization is not yet understood. One aspect of the PGT reaction that is central to defining the mechanism is the direction of glycan chain elongation ( Figure 2A). 2 Here, we describe an approach to test the direction of glycan chain growth. We have applied this strategy to four different PGTs from both Gram-negative and Grampositive organisms, including two PGTs for which crystal structures were recently reported. 3 The results show that all these PGTs polymerize Lipid II by the addition of new disaccharide units to the anomeric (diphospholipid) end of the elongating polymer (called the "reducing end" by convention, although not a lactol).Our strategy to determine the direction of glycan polymerization, illustrated in Figure 2B, relies on the synthesis of nascent peptidoglycan chains that are blocked at their non-reducing ends ( Figure 2B). If elongation occurs by reaction at the reducing end of the growing polymer, PGTs should elongate these substrates in the presence of Lipid II ( Figure 2B NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript right). If elongation occurs in the other direction, then these blocked oligomers will not be substrates for PGTs ( Figure 2B, left).To enable our strategy for probing the direction of chain elongation, we required a facile method to selectively modify the non-reducing ends of the nascent glycan chains. We chose to utilize β-1,4-galactosyltransferase (GalT), an enzyme that transfers galactose (Gal) from UDP-Gal ...

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 is directly involved in the reduction, and R2 contains the diferric-tyrosyl radical (Y⅐) cofactor essential for the initiation of reduction. Saccharomyces cerevisiae has two RNRs; Y1 and Y3 correspond to R1, whereas Y2 and Y4 correspond to R2. Y4 is essential for diferric-Y⅐ formation in Y2 from apoY2, Fe 2؉ , and O2. The actual function of Y4 is controversial. Y2 and Y4 have been further characterized in an effort to understand their respective roles in nucleotide reduction. (His) 6-Y2, Y4, and (His) 6-Y4 are homodimers, isolated largely in apo form. Their CD spectra reveal that they are predominantly helical. The concentrations of Y2 and Y4 in vivo are 0. R ibonucleotide reductases (RNRs) play an essential role in DNA replication and repair by providing all of the monomeric deoxynucleotides required for these processes. The genome sequencing projects have revealed that both prokaryotes and eukaryotes possess multiple RNRs, whose functions remain to be elucidated (1). Class I RNRs are composed of two homodimeric subunits: R1 (␣2) and R2 (␤2). Both subunits are essential for activity. R1 contains the binding sites for the nucleoside diphosphate substrates and the deoxynucleotide and ATP allosteric effectors that govern which nucleotide is reduced and its rate of reduction. R2 possesses a diferric-tyrosyl radical (Y⅐) cofactor essential for initiation of nucleotide reduction on R1 (2, 3).Four genes have been identified that encode RNR subunits in Saccharomyces cerevisiae: RNR1 and RNR3 (their gene products designated Y1 and Y3) are R1 homologues, and RNR2 and RNR4 (their gene products designated Y2 and Y4) are R2 homologues (4-9). Y1 and Y3 share 80% sequence identity (4). RNR1 expression is cell cycle regulated, and the gene is essential for mitotic viability (5, 10). In contrast, RNR3 is not expressed under normal growth conditions. Transcription of RNR1 and RNR3 is inducible by DNA damage; the mRNA of the latter is up-regulated Ͼ500-fold (5). Y2 and Y4 share 56% sequence identity. Y4 contains several unusual features relative to a canonical R2. It lacks about 50 amino acid residues from the N terminus, and of the 16 amino acid residues conserved in almost all class I R2s, six have been replaced in Y4. The most notable substitutions are two histidines and a glutamate, ligands of the di-iron center, which have been replaced by two tyrosines and an arginine. Such substitutions would be expected to disrupt the ability of Y4 to bind iron. RNR2 is essential for mitotic viability (10,11). RNR4 also appears to be important for mitotic viability, but its essentiality varies with genetic background (8, 9, 12). In the one case where a Y4 knockout was shown to be lethal, the lethality could be suppressed by overexpression of Y1 and Y3 (8).The transcriptional regulation of the yeast RNR genes has been studied extensively. However, little is known about the biochem...

Background: Nbp35 and Cfd1 are iron-sulfur cluster scaffolds with an NTPase domain of unknown function. Results: Nucleotide binding and hydrolysis assays paired with mutagenesis demonstrate ATP hydrolysis by these cluster scaffolds. Conclusion: Nbp35 and the Nbp35-Cfd1 complex are ATPases. Significance: This first demonstration of ATPase activity enables future investigation of how nucleotide influences cluster biogenesis by this large family of proteins.

The class I ribonucleotide reductases (RNRs) are composed of two homodimeric subunits: R1 and R2. R2 houses a diferric-tyrosyl radical (Y•) cofactor. Saccharomyces cerevisiae has two R2s: Y2 (β 2 ) and Y4 (β′ 2 ). Y4 is an unusual R2 because three residues required for iron binding have been mutated. While the heterodimer (ββ′) is thought to be the active form, several rnr4Δ strains are viable. To resolve this paradox, N-terminally epitope-tagged β and β′ were expressed in E. coli or integrated into the yeast genome. In vitro exchange studies reveal that when apo-(His 6 )-β 2 ( His β 2 ) is mixed with β′ 2 , apo-His ββ′ forms quantitatively within 2 min. In contrast, holo-ββ′ fails to exchange with apo-His β 2 to form holo-His ββ and β′ 2 . Isolation of genomically encoded tagged β or β′ from yeast extracts gave a 1:1 complex of β and β′, suggesting that ββ′ is the active form. The catalytic activity, protein concentrations, and Y• content of the rnr4Δ and wild type (wt) strains were compared to clarify the role of β′ in vivo. The Y• content of rnr4Δ is 15-fold less than that of wt, consistent with the observed low activity of rnr4Δ extracts (<0.01 nmol min −1 mg −1 ) versus wt (0.06 ± 0.01 nmol min −1 mg −1 ). FLAG β 2 isolated from the rnr4Δ strain has a specific activity of 2 nmol min −1 mg −1 , similar to that of reconstituted apo-His β 2 (10 nmol min −1 mg −1 ), but significantly less than holo-His ββ′ (~2000 nmol min −1 mg −1 ). These studies together demonstrate that β′ plays a crucial role in cluster assembly in vitro and in vivo and that the active form of the yeast R2 is ββ′.Ribonucleotide reductases (RNRs) 1 catalyze the conversion of ribonucleotides to deoxyribonucleotides, providing the monomeric precursors for DNA replication and repair (1). The class I RNRs are composed of a large subunit, R1, and a small subunit, R2. R1 contains the site of nucleotide reduction and the allosteric effector binding sites that control the rate and the specificity of nucleotide reduction. R2 houses the diferric-tyrosyl radical † D.L.P. and A.D.O. were supported in part by the NIH training grant 5T32 CA 09112-28. J.S. acknowledges support of the NIH (GM29595). M.H. acknowledges support of the NIH (CA095207) and the ACS (0305001GMC).

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two types of subunits: RNR1 contains the active site for reduction and the binding sites for the nucleotide allosteric effectors. RNR2 contains the diiron-tyrosyl radical (Y⅐) cofactor essential for the reduction process. Studies in yeast have recently identified four RNR subunits: Y1 and Y3, Y2 and Y4. These proteins have been expressed in Saccharomyces cerevisiae and in Escherichia coli and purified to Ϸ90% homogeneity. The specific activity of Y1 isolated from yeast and E. coli is 0.03 mol⅐min ؊1 ⅐mg ؊1 and of (His)6-Y2 Ribonucleotide reductases (RNRs) in all organisms catalyze the reduction of nucleotides to deoxynucleotides, an essential step in DNA biosynthesis. These enzymes play a central role in maintaining a balanced pool of cellular deoxynucleotides required for fidelity of DNA replication and repair (1-3). The aerobic Escherichia coli RNR is the prototype for class I RNRs found in all eukaryotes and most prokaryotes. The class I RNRs are composed of two homodimeric proteins: RNR1 (␣ 2 , 171 kDa) and RNR2 (␤ 2 , 87 kDa). RNR1 contains the site where NDPs are reduced and the sites for the allosteric effectors that govern activity and specificity. RNR2 contains a diiron clustertyrosyl radical (Y⅐) cofactor required for nucleotide reduction activity (4).The mechanism by which the diiron-Y⅐ cofactor is generated in vitro has been studied extensively (5, 6). However, the mechanism by which the cluster is assembled in vivo has remained largely unexplored. The completion of the Saccharomyces cerevisiae genome sequencing project and the identification of a large number of yeast mutants involved in iron homeostasis (7,8) have suggested that yeast may be an excellent system to investigate diiron-Y⅐ cofactor assembly and its relationship to nucleotide reduction in vivo.Recent studies have identified four genes encoding RNR subunits in S. cerevisiae: RNR1 and RNR3 (designated Y1 and Y3) and RNR2 and RNR4 (designated Y2 and Y4) (9-13). Y1 and Y3, analogous to RNR1 of E. coli RNR, share Ϸ80% sequence identity. Y1 expression is cell cycle-regulated, and the gene is essential for mitotic viability. In contrast, Y3 is not expressed under normal vegetative conditions. However, the presence of high copy numbers of the Y3 gene has been shown to suppress a lethal mutation in the Y1 gene, suggesting that Y3 encodes a functional protein (11). Transcription of Y1 and Y3 genes is inducible by DNA damage, the latter 100-fold (11). The Y2 gene encodes a protein analogous to E. coli RNR2 and is essential for mitotic viability. The Y4 gene encodes a protein sharing 56% sequence identity with Y2. Y4, however, contains several unusual features not found in Y2 and mammalian RNR2s. These features include a deletion of 51 aa residues at its N terminus and a substitution of 6 of the 16 aa residues conserved in all class I RNR2s (12, 13). The most notable substitutions are the replacement of two histidines and one g...