In the Neurospora VS ribozyme, magnesium ions facilitate formation of a loop-loop interaction between stem-loops I and V, which is important for recognition and activation of the stem-loop I substrate. Here, we present the high-resolution NMR structure of stem-loop V (SL5) in the presence of Mg(2+) (SL5(Mg)) and demonstrate that Mg(2+) induces a conformational change in which the SL5 loop adopts a compact structure with most characteristics of canonical U-turn structures. Divalent cation-binding sites were probed with Mn(2+)-induced paramagnetic line broadening and intermolecular NOEs to Co(NH(3))(6)(3+). Structural modeling of Mn(H(2)O)(6)(2+) in SL5(Mg) revealed four divalent cation-binding sites in the loop. Sites 1, 3, and 4 are located in the major groove near multiple phosphate groups, whereas site 2 is adjacent to N7 of G697 and N7 of A698 in the minor groove. Cation-binding sites equivalent to sites 1-3 in SL5 are present in other U-turn motifs, and these metal-binding sites may represent a common feature of the U-turn fold. Although magnesium ions affect the loop conformation, they do not significantly change the conformation of residues 697-699 involved in the proposed Watson-Crick base pairs with stem-loop I. In both the presence and the absence of Mg(2+), G697, A698, and C699 adopt an A-form structure that exposes their Watson-Crick faces, and this is compatible with their proposed interaction with stem-loop I. In SL5(Mg), however, U700 becomes exposed on the minor groove face of the loop in the proximity of the bases of G697, A698, and C699, suggesting that the Mg(2+)-bound conformation of stem-loop V allows additional contacts with stem-loop I. These studies improve our understanding of the role of Mg(2+) in U-turn structures and in substrate recognition by the VS ribozyme.

Deoxycytidine kinase (dCK) is a rate-limiting enzyme in deoxyribonucleoside salvage, a metabolic pathway that recycles products of DNA degradation. dCK phosphorylates and therefore activates nucleoside analog prodrugs frequently used in cancer, autoimmunity, and viral infections. In contrast to its well established therapeutic relevance, the biological function of dCK remains enigmatic. Highest levels of dCK expression are found in thymus and bone marrow, indicating a possible role in lymphopoiesis. To test this hypothesis we generated and analyzed dCK knockout (KO) mice. dCK inactivation selectively and profoundly affected T and B cell development. A 90-fold decrease in thymic cellularity was observed in the dCK KO mice relative to wild-type littermates. Lymphocyte numbers in the dCK KO mice were 5-to 13-fold below normal values. The severe impact of dCK inactivation on lymphopoiesis was unexpected given that nucleoside salvage has been thought to play a limited, "fine-tuning" role in regulating deoxyribonucleotide triphosphate pools produced by the de novo pathway. The dCK KO phenotype challenges this view and indicates that, in contrast to the great majority of other somatic cells, normal lymphocyte development critically requires the deoxyribonucleoside salvage pathway.

An important step in the substrate recognition of the Neurospora Varkud Satellite (VS) ribozyme is the formation of a magnesium-dependent loop/loop interaction between the terminal loops of stem-loops I and V. We have studied the structure of stem-loop V by nuclear magnetic resonance spectroscopy and shown that it adopts a U-turn conformation, a common motif found in RNA. Structural comparisons indicate that the U-turn of stem-loop V fulfills some but not all of the structural characteristics found in canonical U-turn structures. This U-turn conformation exposes the Watson-Crick faces of the bases within stem-loop V (G697, A698, and C699) and makes them accessible for interaction with stem-loop I. Using chemical-shift mapping, we show that magnesium ions interact with the loop of the isolated stem-loop V and induce a conformational change that may be important for interaction with stem-loop I. This study expands our understanding of the role of U-turn motifs in RNA structure and function and provides insights into the mechanism of substrate recognition in the VS ribozyme.

Endogenous thymidine plays a critical role in the induction of replication stress in thymocytes.

Many virulence factors in GramSurface proteins on bacteria are frequently virulence factors, promoting bacterial adhesion, resistance to phagocytic killing, and host cell invasion during infection. In Gram-positive bacteria these proteins are often covalently anchored to the cell wall by sortase enzymes, a family of novel cysteine transpeptidases (1-3). The sortase A protein (SrtA) 2 from Staphylococcus aureus has been characterized extensively (4) and anchors proteins bearing a cell wall sorting signal that consists of a conserved LPXTG motif (where X is any amino acid), a hydrophobic domain, and a tail of mostly positively charged residues (4 -6). SrtA cleaves in between the threonine and glycine of the LPXTG motif (7) and catalyzes the formation of a peptide bond between the carboxylgroup of the threonine and the amine-group of the cell-wall precursor lipid II (7-9). The lipid II-linked protein is then incorporated into the peptidoglycan of the cell wall via the transglycosylation and transpeptidation reactions of bacterial cell-wall synthesis. Sortases represent an attractive target for new anti-infective agents, because they are widely distributed among a variety of bacterial pathogens (10, 11) (e.g. Bacillus anthracis, Listeria monocytogenes, Streptococcus pneumoniae, and Streptococcus pyogenes), and have been shown to be required for virulence (12-16).The catalytic domain of SrtA (SrtA ⌬N59 , residues 60 -206) adopts a conserved eight-stranded ␤-barrel fold (17, 18). The active site is organized around the catalytically essential side chain of Cys-184, whose thiolate nucleophilically attacks the threonine carbonyl carbon within the LPXTG sorting signal, forming a thioester linkage between the enzyme and substrate (19). In addition to Cys-184, the hydrophilic side chains of His-120 and Arg-197 are absolutely required for catalysis (20 -22). These residues likely participate in general acid/base catalysis, and one of them must activate the thiol for nucleophilic attack, because it is protonated at neutral pH (23). The indole ring of Trp-194 partially shields the cysteine thiol from the solvent, and its mutation to alanine reduces enzyme activity 4-fold through an unknown mechanism (20). Using NMR and crystallography, the LPXTG sorting signal binding site has recently been localized to a surface formed by strands ␤4 and ␤7, and to a proximal loop that connects strands ␤6 to ␤7(the ␤6/␤7 loop) (18,22). Substrate binding may occur through an induced-fit mechanism involving conformational changes in the ␤6/␤7 loop, because it is disordered in the absence of the sorting signal substrate (17,18). Ca 2ϩ stimulates the activity of SrtA ⌬N59 in vitro (17) and may enable S. aureus to increase the rate of surface protein anchoring as it encounters elevated concentrations of this ion at sites of infection. Because many surface proteins function as virulence factors, the stimulatory effect of Ca 2ϩ likely plays an important role in the infection process. Previously we showed that Ca 2ϩ bound to an ordered pocket positio...

Positron emission tomography (PET) imaging reporter genes (IRGs) and PET reporter probes (PRPs) are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials.

Background: Humanized PET reporter gene (PRG) systems are needed to replace immunogenic, viral-derived systems. Results: Employing a structure-guided approach, we developed a highly sensitive humanized PRG characterized by reduced activity for its natural substrates. Conclusion: Sensitivity of PRGs can be improved by reducing their endogenous activities. Significance: Our method can be employed to rapidly develop highly sensitive humanized PRGs.

Gemcitabine (2 ,2 -difluorodeoxycytidine, dFdC) and cytosine arabinoside (cytarabine, ara-C) represent a class of nucleoside analogs used in cancer chemotherapy. Administered as prodrugs, dFdC and ara-C are transported across cell membranes and are converted to cytotoxic derivatives through consecutive phosphorylation steps catalyzed by endogenous nucleoside kinases. Deoxycytidine kinase (DCK) controls the rate-limiting step in the activation cascade of dFdC and ara-C. DCK activity varies significantly among individuals and across different tumor types and is a critical determinant of tumor responses to these prodrugs. Current assays to measure DCK expression and activity require biopsy samples and are prone to sampling errors. Noninvasive methods that can detect DCK activity in tumor lesions throughout the body could circumvent these limitations. Here, we demonstrate an approach to detecting DCK activity in vivo by using positron emission tomography (PET) and 18