RESUMEN
Trypanosoma brucei encodes a protein (denoted TbABH) that is homologous to AlkB of Escherichia coli and AlkB homolog (ABH) proteins in other organisms, raising the possibility that trypanosomes catalyze oxidative repair of alkylation-damaged DNA. TbABH was cloned and expressed in E. coli, and the recombinant protein was purified and characterized. Incubation of anaerobic TbABH with Fe(II) and α-ketoglutarate (αKG) produces a characteristic metal-to-ligand charge-transfer chromophore, confirming its membership in the Fe(II)/αKG dioxygenase superfamily. The protein binds to DNA, with a clear preference for alkylated oligonucleotides according to results derived by electrophoretic mobility shift assays. Finally, the protozoan gene was shown to partially complement E. coli alkB cells when stressed with methylmethanesulfonate; thus confirming assignment of TbABH as a functional AlkB protein in T. brucei.
Asunto(s)
Reparación del ADN/fisiología , ADN Protozoario/genética , Proteínas Protozoarias/fisiología , Trypanosoma brucei brucei/genética , Alquilación , Secuencia de Aminoácidos , Cromatografía en Gel , Clonación Molecular , ADN Protozoario/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli , Expresión Génica , Prueba de Complementación Genética , Humanos , Datos de Secuencia Molecular , Oxidación-Reducción , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Espectrofotometría Ultravioleta , Trypanosoma brucei brucei/químicaRESUMEN
INTRODUCTION: MedEdPORTAL is an open-access journal for health professions educators to publish their educational activities. The Educational Summary Report (ESR) is the manuscript that represents scholarly expression of those activities, aligned with Glassick's criteria for scholarship; however, prospective authors face challenges in writing ESRs, which can lead to rejection. METHODS: We developed a conference workshop to teach health professions educators how to write an ESR by reviewing a sample ESR in small groups. The workshop began with a didactic on best practices in crafting each section of an ESR. We then divided participants into small groups to review an assigned section of a sample ESR using a reviewer's checklist and completing a templated flip chart. Each small group then reported out in a large-group discussion. A conference evaluation was distributed online to solicit perceptions of the workshop's effectiveness. RESULTS: The 90-minute workshop was presented by separate teams of two facilitators at three national conferences. Approximately 35 participants attended the first workshop, and 50 attended the second and third workshops. Survey feedback from 19 respondents (38%) to the evaluation survey at the third workshop was representative of the previous two iterations and demonstrated that workshop content and materials were helpful. DISCUSSION: A workshop enabling educators to serve as group peer reviewers of a sample ESR for a MedEdPORTAL submission was well received. Associate editors, faculty mentors, and other experienced faculty development leaders can use these materials to support future authors in submitting to MedEdPORTAL while providing opportunities for national presentations.
Asunto(s)
Educación Médica , Informe de Investigación , Becas , Humanos , Estudios Prospectivos , EdiciónRESUMEN
Two genes from Trypanosoma brucei brucei are predicted to encode Fe(II)- and alpha-ketoglutarate-dependent enzymes related to fungal thymine 7-hydroxylase. Transcription of the thymine hydroxylase-like genes is up-regulated in the bloodstream form of the parasite over the insect form, whereas Western blot analysis indicates more cross-reactive protein in the latter life stage. The genes were cloned, the proteins purified from Escherichia coli, and both proteins were shown to bind Fe(II) and alpha-ketoglutarate, confirming proper folding. The isolated proteins were incubated with Fe(II)- and alpha-ketoglutarate plus thymine, thymidine, and other putative substrates, but no activity was detected. Furthermore, no thymine 7-hydroxylase activity was detected in extracts of procyclic or bloodstream form cells. Although the functions of these proteins remain unknown, we conclude they are unlikely to be involved in thymine salvage.
Asunto(s)
Oxigenasas de Función Mixta/genética , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/inmunología , Western Blotting , Escherichia coli/genética , Cromatografía de Gases y Espectrometría de Masas , Regulación Enzimológica de la Expresión Génica , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/genética , Oxigenasas de Función Mixta/biosíntesis , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/inmunología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Conejos , Rhodotorula/enzimología , Rhodotorula/genética , Espectrofotometría Ultravioleta , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/inmunologíaRESUMEN
Trypanosomatids contain an unusual DNA base J (beta-d-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe(2+) and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe(2+) and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Glucósidos/biosíntesis , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Uracilo/análogos & derivados , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Proteínas de Unión al ADN/clasificación , Dioxigenasas/clasificación , Glucósidos/química , Glucósidos/metabolismo , Leishmania/genética , Oxigenasas de Función Mixta/clasificación , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Protozoarias/clasificación , Uracilo/biosíntesis , Uracilo/química , Uracilo/metabolismoRESUMEN
Fe(II)/alpha-ketoglutarate-dependent hydroxylases uniformly possess a double-stranded beta-helix fold with two conserved histidines and one carboxylate coordinating their mononuclear ferrous ions. Oxidative decomposition of the alpha-keto acid is proposed to generate a ferryl-oxo intermediate capable of hydroxylating unactivated carbon atoms in a myriad of substrates. This Perspective focuses on a subgroup of these enzymes that are involved in pyrimidine salvage, purine decomposition, nucleoside and nucleotide hydroxylation, DNA/RNA repair, and chromatin modification. The varied reaction schemes are presented, and selected structural and kinetic information is summarized.