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Authors Associated with MMG:
Ian Odell – recently earned his Ph.D. in MMG. At present, Ian is finishing clinical rotations for his M.D. in 2012, and interviewing for residency training programs.
Joy-El Barbour – conducted undergraduate research and worked as a technician in the MMG Department. She is now a Ph.D. candidate at U.C.Berkeley.
Joann Sweasy – Adjunct professor in MMG and a Professor in the Department of
Therapeutic Radiology at Yale.
Susan Wallace – Professor and Chair of MMG.
David Pederson – Associate Professor and Director of the Graduate Program in
MMG.

Summary:
The eukaryotic enzymes that repair DNA damage must function in presence of chromatin. To study the effects of chromatin on DNA repair, the authors reconstituted nucleosomes containing oxidatively damaged DNA. They then reconstituted the entire four-step DNA repair reaction, using purified, recombinant enzymes. The authors demonstrated that the DNA glycosylase hNTH1 can recognize and excise damaged bases from nucleosomes without irreversibly disrupting the nucleosome. The same proved true for apurinic endonuclease APE (which was observed to displace hNTH1 from its product) and, to a lesser extent, the DNA repair polymerase Pol Beta. However, Ligase IIIa, which catalyzes the final step in repair, disrupted nucleosomes when added along with the scaffolding protein XRCC1. Importantly, the Ligase IIIa-XRCC1 heterodimer proved able to disrupt nucleosomes containing either Pol Beta or ligase IIIa substrates. Because Pol Beta, ligase IIIa and XRCC1 form a trimeric complex, these results suggest that, in vivo, the first two steps in the repair of oxidative damage occur in nucleosomes, but that nucleosome disruption facilitates the final two steps in repair.

Impact and Significance:
Each day, approximately 20,000 oxidative lesions form in the DNA of every nucleated human cell. The authors’ findings provide insights into rate-limiting steps that govern DNA damage repair in chromatin and reveal a unique role for ligase IIIα-XRCC1 in enhancing the repair efficiency in the presence of nucleosomes.

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Authors Associated with MMG:
Whittney Dotzler Barkhuff – was an MD/PhD student in the CMB program
Stacey Gilk – was an MMG graduate student
Luke Tilley – is an MMG graduate student
Gary Ward – is a Professor in the MMG Department

Summary:
The inner membrane complex (IMC) is thought to be important for parasite shape, motility, and replication and is comprised of a series of flattened vesicles at the periphery of apicomplexan parasites. The authors had previously identified the protein TgPhlL1 as a component of the IMC concentrated at the apical end of the Toxoplasma gondii parasite, but the function remained unknown. Here the authors generated TgPhlL1 deletion mutants and have shown an altered morphology that can be rescued with the wild-type allele. The TgPhlL1 mutants compete poorly with wild-type in culture and the mutants have reduced virulence in a mouse infection model.

Impact and Significance:
These findings demonstrate a role for TgPhIL1 in the morphology, growth and fitness of T. gondii. Based on these findings, TgPhlL1 may represent a novel therapeutic target for the treatment of toxoplasmosis.

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Authors Associated with MMG:
Liam F. Fitzsimmons – is a Research Technician in MMG
I. Neil Sarkar – is an Assistant Professor in MMG
Matthew J. Wargo – is an Assistant Professor in MMG

Summary:
The authors sought to generate choline analogues to study bacterial choline catabolism in vitro and in situ. Here they report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of dimethylglycine demethylation in Pseudomonas aeruginosa. Using genetic analyses and ¹³C nuclear magnetic resonance they demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine, a conclusion supported by the function of chemically synthesized propargylmethylglycine. Because the enzyme targeted by propargylmethylglycine has homologues in many bacteria, the authors examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. While propargylcholine only inhibited growth in P. aeruginosa, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, including Pseudomonas mendocina, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Burkholderia ambifaria, and Sinorhizobium meliloti.

Impact and Significance:
Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Likewise, aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. To generate a set of tools to study choline catabolism in vitro and in situ, the authors synthesized a set of inhibitory molecules that targeted a specific step in the choline catabolic pathway. These compounds affected P. aeruginosa virulence-related phenotypes and propargylmethylglycine prevented growth on choline in a variety of proteobacteria. Thus the authors conclude that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolism in situ.

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Authors’ Associations:
Karl E. Zahn – is a graduate student in the CMB program
Sylvie Doublié – is a Professor in the MMG Department
Matthias Götte and Egor Tchesnokov are collaborators from McGill University

Summary:
In this paper, the authors took advantage of a chimeric version of the bacteriophage RB69 DNA polymerase that retained the phosphonoformic acid (PFA) sensitivity of the human cytomegalovirus (HCMV) DNA polymerase but was expressed at levels high enough to pursue structural studies. By comparing the crystal structures of this chimeric polymerase with and without PFA, the authors were able to show that PFA binds to the active site and interacts with two conserved basic residues known to contact the triphosphate tail of incoming nucleotides. The drug also was observed chelating one of the metal ions required for the catalysis by this enzyme. These findings provide a model for the mechanism by which PFA inhibits viral polymerases.

Impact and Significance:
The authors show that PFA inhibits the HCMV chimeric polymerase by trapping the closed, untranslocated form of the polymerase complexed to DNA. Mutational analysis demonstrated that an inter-domain clash biases the chimera toward the closed conformation. This is likely key in the acquired PFA sensitivity and the chimera might aid future drug design targeting HCMV polymerase.

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Authors’ Association with MMG:
Krista Brooks – is a recent MMG graduate student now a postdoc at Scripps
Ken Hampel – is an Assistant Professor in the MMG Department

Summary:
Riboswitches are regions of bacterial and eukaryotic mRNAs that function as metabolite-sensing genetic switches. The glmS ribozyme is a broadly distributed Gram-positive bacterial riboswitch that senses the intercellular concentration of glucosamine 6-phosphate. Upon binding this metabolite the glmS ribozyme catalyzes a site-specific RNA cleavage within the 5’UTR of the glmS mRNA. Cleavage of the 5’ UTR exposes a 5’ hydroxyl-terminated mRNA to the rapid degradation by 5’-3’ RNases. The result of the action of this RNA switch, therefore, is to mediate feedback repression of the GlmS protein, L-glutamine:D-fructose-6-phosphate aminotransferase. The ligand binding specificity is therefore central to the function of the riboswitch.

The requirements for rapid binding of the ligand were probed by kinetic measurements of the cleavage reaction under conditions where native folding of the RNA, which is slow and rate-limiting for the reaction (Brooks and Hampel, 2009), could be carried out as a preliminary step. Our data indicate that rapid ligand binding and catalysis requires divalent metal ions and that this requirement is linked to the presence of the phosphate moiety of the ligand. The absence of either Mg2+ or the ligand phosphate result in identical, non-additive losses in catalytic activity. In addition, we show that the glmS ribozyme is able to fold into its native conformation in the presence of high concentrations of monovalent cations. Our data therefore show no obligatory role for divalent cations outside of the ligand binding pocket.

Impact and Significance:
The GlmS enzyme catalyzes the rate-limiting step in the production of the UDP-N-acetylglucosamine (UDP-GlcNAc), a precursor of cell wall constiuents peptidoglycan and lipopolysaccharides, in Gram negative and positive bacteria. This pathway is the target of several established antibiotics and continues to be an attractive target for new antibiotic research. Several important Gram positive bacterial human pathogens utilize the glmS riboswitch to control this pathway, including S. aureus, Listeria momocytogenese, and B. anthracis. Our data may suggest new approaches to the development of antibiotics which target this genetic switch.

Our data also impact our understanding of the biochemistry of bacterial riboswitches. The natural ligand of the glmS ribozyme, GlcN6P, is believed to be a coenzyme in the site-specific RNA cleavage reaction. Since RNA has a limited enzymatic repertoire it has been speculated that one way in which RNAs could have carried out a broad range of metabolic chemistry in a hypothesized “RNA world” is to utilize coenzymes. Indeed, many bacterial riboswitches bind protein coenzymes such as S-adenosyl methionine, flavin mononucleotide and cobalamin. However, only the glmS ribozyme has been shown to use a coenzyme in the mechanistic reaction chemistry. Our data demonstrate essential coenzyme binding requirements for the glmS ribozyme.

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Authors’ Association with MMG:
Pam Lescault – is an MMG Graduate Student
Ann Thompson – is an MMG Graduate Student
Veerupaxagouda Patil – is a former post-doc in Matrajt Lab
Dario Lirussi – is an MMG Graduate Student
Amanda Burton – is a former MMG Undergraduate Student
Juan Margarit – is a former technician in Matrajt Lab
Jeffrey Bond – is an MMG Associate Professor
Marianna Matrajt – is an MMG Assistant Professor

Summary:
A critical portion of the lifecycle of the parasite Toxoplasma gondii is the invasion of host cells followed by replication of the tachyzoites and development of the parasite into the bradyzoite stage. Bradyzoites are a latent stage that can reside for long periods of time in host tissues and can be reactivated to generate fatal encephalitis in immune-compromised patients. Lescault and colleagues used microarray analysis of wild-type and mutant T. gondii to identify genes involved in the tachyzoite to bradyzoite transition. During this analysis they identified a novel extracellular tachyzoite stage that may be important for reinvasion of host cells during the acute phase of infection.

Impact and Significance:
T. gondii is an intracellular parasite of mammals whose primary host is the domestic cat. It is readily passed to humans who typically carry the parasite asymptomatically as intracellular bradyzoites. While bradyzoites rarely cause symptoms in healthy individuals, those with immune-deficiencies including people with AIDS and those undergoing chemotherapy are susceptible to potentially fatal toxoplasmosis. Understanding the gene regulation and proteins involved in the switch between bradyzoite and tachyzoite will be critical to identification of new therapeutic targets.