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 ...