RESUMEN
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
Synthesis of the modified thymine base, beta-d-glucosyl-hydroxymethyluracil or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream form specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. In order to analyze the function of base J in the regulation of antigenic variation, we are characterizing the regulatory mechanism of J biosynthesis. We have recently proposed a model in which chromatin remodeling by a SWI2/SNF2-like protein (JBP2) regulates the developmental and de novo site-specific localization of J synthesis within bloodstream form trypanosome DNA. Consistent with this model, we now show that JBP2 (-/-) bloodstream form trypanosomes contain five-fold less base J and are unable to stimulate de novo J synthesis in newly generated telomeric arrays.
Asunto(s)
Sangre/parasitología , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Glucósidos/metabolismo , Telómero/metabolismo , Trypanosoma brucei brucei/metabolismo , Uracilo/análogos & derivados , Animales , Variación Antigénica , ADN Protozoario/metabolismo , Proteínas de Unión al ADN/genética , Glicosilación , Telómero/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Uracilo/metabolismoRESUMEN
Synthesis of the modified thymine base beta-D-glucosyl-hydroxymethyluracil, or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream-form-specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. The mechanism of developmental and telomeric-specific regulation of J synthesis is unknown. We have previously identified a J binding protein (JBP1) involved in propagating J synthesis. We have now identified a homolog of JBP1, JBP2, containing a domain related to the SWI2/SNF2 family of chromatin remodeling proteins that is upregulated in bloodstream form cells and interacts with nuclear chromatin. We show that expression of JBP2 in procyclic form cells leads to de novo J synthesis within telomeric regions of the chromosome and that this activity is inhibited after mutagenesis of conserved residues critical for SWI2/SNF2 function. We propose a model in which chromatin remodeling by JBP2 regulates the initial sites of J synthesis within bloodstream form trypanosome DNA, with further propagation and maintenance of J by JBP1.