Biosynthesis and Function of the Modified DNA Base β-d-Glucosyl-Hydroxymethyluracil in Trypanosoma brucei

Abstract

β-d-Glucosyl-hydroxymethyluracil, also called J, is a modified DNA base conserved among kinetoplastid flagellates. InTrypanosoma brucei, the majority of J is present in repetitive DNA but the partial replacement of thymine by J also correlates with transcriptional repression of the variant surface glycoprotein (VSG) genes in the telomeric VSG gene expression sites. To gain a better understanding of the function of J, we studied its biosynthesis in T. brucei and found that it is made in two steps. In the first step, thymine in DNA is converted into hydroxymethyluracil by an enzyme that recognizes specific DNA sequences and/or structures. In the second step, hydroxymethyluracil is glucosylated by an enzyme that shows no obvious sequence specificity. We identified analogs of thymidine that affect the J content of theT. brucei genome upon incorporation into DNA. These analogs were used to study the function of J in the control of VSG gene expression sites. We found that incorporation of bromodeoxyuridine resulted in a 12-fold decrease in J content and caused a partial derepression of silent VSG gene expression site promoters, suggesting that J might strengthen transcriptional repression. Incorporation of hydroxymethyldeoxyuridine, resulting in a 15-fold increase in the J content, caused a reduction in the occurrence of chromosome breakage events sometimes associated with transcriptional switching between VSG gene expression sites in vitro. We speculate that these effects are mediated by the packaging of J-containing DNA into a condensed chromatin structure.

Bloodstream Form-specific Up-regulation of Silent VSG Expression Sites and Procyclin in Trypanosoma brucei after Inhibition of DNA Synthesis or DNA Damage

“…ES derepression was interpreted as a consequence of reduction in J, as incubation with BrdUrd resulted in a 12-fold decrease in the levels of J. However, overproduction of J by incubation with hydroxymethyldeoxyuridine also resulted in derepression of silent sites, making it possible that perturbance of the DNA structure was the critical factor rather than the amount of J itself (65). Although incubation of our T. brucei GFP reporter strain with up to 400 M BrdUrd resulted in silent ES upregulation, levels were not as striking as with the other treatments shown here (results not shown).…”

FeII/α-ketoglutarate hydroxylases involved in nucleobase, nucleoside, nucleotide, and chromatin metabolism

“…This reaction is not specific to the sequence or life cycle-stage as demonstrated by the random placement of base J in the bloodstream and procyclic forms of cells grown with HOMedU included in the medium. 43 A J binding protein (JBP1) was identified in T. brucei and shown to specifically bind to base J in DNA. 49 Subsequently, another trypanosomal protein was discovered to share some N-terminal sequence identity with JBP1 (along with exhibiting homology to the SWI2/SNF2 chromatin remodeling protein family) and named JBP2 even though it does not bind to J-containing DNA.…”

“…41,42 The modified base is thought to induce chromatin compaction and thereby stabilize the chromosome from rearrangements within these repetitive sequences. 43,44 Moreover, base J is suggested to play a role in gene silencing related to the process of antigenic variation. 51 Mutation of putative active-site residues in JBP1 yielded protein that was unable to complement a JBP1 null mutant to restore base J levels.…”

JBP2, a SWI2/SNF2-like protein, regulates de novo telomeric DNA glycosylation in bloodstream form Trypanosoma brucei

“…Only one of the 20 expression sites is active at any given time. The presence of J within the ~19 inactive telomeric VSG gene expression sites but not in the active site suggests that J may be involved in the transcriptional repression of VSG gene expression sites and thus, antigenic variation [3,5,6].…”

“…In order to analyze the biological function of base J in T. brucei, we need to first characterize the factor(s) regulating Jbiosynthesis. Substantial indirect evidence [6][7][8] indicates that the modification of thymine in trypanosome DNA occurs in two steps: a thymine is oxidized to hydroxymethyluracil (HOMeUra) by a putative thymine-7-hydroxylase (TH), followed by glucosylation of the new base by a putative glucosyltransferase (GT). All current data suggests that the stage specific regulation occurs at the first step, since procyclic-form trypanosomes are able to synthesize J when grown in the presence of hydroxymethyldeoxyuridine (HOMedU), the intermediate in the J-biosysnthesis pathway.…”