β- d -Glucosyl-hydroxymethyluracil is a conserved DNA modification in kinetoplastid protozoans and is abundant in their telomeres

Abstract

A BSTR ACTThe unusual DNA base ␤-D-glucosylhydroxymethyluracil, called ''J,'' replaces Ϸ0.5-1% of Thy in DNA of African trypanosomes but has not been found in other organisms thus far. In Trypanosoma brucei, J is located predominantly in repetitive DNA, and its presence correlates with the silencing of telomeric genes. Using antibodies specific for J, we have developed sensitive assays to screen for J in a range of organisms and have found that J is not limited to trypanosomes that undergo antigenic variation but is conserved among Kinetoplastida. In all kinetoplastids tested, including the human pathogens Leishmania donovani and Trypanosoma cruzi, J was found to be abundantly present in the (GGGTTA) n telomere repeats. Outside Kinetoplastida, J was found only in Diplonema, a small phagotrophic marine f lagellate, in which we also identified 5-MeCyt. Fractionation of Diplonema DNA showed that the two modifications are present in a common genome compartment, which suggests that they may have a similar function. Dinof lagellates appear to contain small amounts of modified bases that may be analogs of J. The evolutionary conservation of J in kinetoplastid protozoans suggests that it has a general function, repression of transcription or recombination, or a combination of both. T. brucei may have recruited J for the control of genes involved in antigenic variation.In the nuclear DNA of Trypanosoma brucei, Ϸ0.5-1% of Thy is replaced by the modified base ␤-D-glucosyl-hydroxymethyluracil (␤-gluc-HOMeUra) (1). This base that we call ''J'' was detected initially by 32 P-nucleotide postlabeling combined with twodimensional TLC (2D-TLC) (2), and we used this technique to show that approximately one-half of the cellular J is present in both strands of the telomeric (GGGTTA) n repeats (3). To map the location of J more precisely, we have generated antisera that immunoprecipitate J-containing duplex DNA and that detect this DNA with high sensitivity and specificity on dot blots (4). We have used these antisera to demonstrate that J is present in other repetitive DNA sequences but not in housekeeping genes or transcribed repeats (4). Moreover, we have shown that J is responsible for the blocked restriction sites that are present in silent telomeric variant surface glycoprotein (VSG) genes but not in actively transcribed VSG genes (4-6). This result has linked J to the transcriptional control of VSG genes.Thus far, J has been detected only in African trypanosome species that undergo antigenic variation (2). The availability of antibodies acting against J has prompted us to reinvestigate whether J is also present in other organisms. With anti-J-DNA immunoblots, approximately one J per 10 7 bases can be detected, which is Ϸ1,000-fold more sensitive than MATERIALS AND METHODSCells and DNA Analysis. DNA was derived from: T. brucei brucei (427); Trypanosoma congolense (WG81 and TSW13 bloodstream forms and WG81 procyclics); Trypanosoma vivax (Y58); Crithidia fasciculata (ϭ C. luciliae); Leishmania donovani (HU3); Leishmania tarentol...

Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome

“…1A. The specific localization of J within the kinetoplastid genome provides the strongest evidence that thymidine residues are modified in DNA rather than being synthesized and then incorporated during DNA replication (2,4). In contrast, when trypanosomes are grown in the presence of hmU, it is incorporated randomly into DNA and then converted to J.…”

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

“…Up to one percent of the thymine in the telomeric and subtelomeric repeats of nuclear DNA in the bloodstream form of T. brucei is replaced with β-D-glucosyl-hydroxymethyluracil (base J) but no modification is found in the insect form of the parasite where antigenic variation is absent. 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.…”

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].…”

“…This hyper-modified base consists of the attachment of a bulky glucose moiety to the thymine base such that it extends into the major groove of the DNA helix. While J is abundantly present in telomeric repeats of all kinetoplastids, in the parasite Trypanosoma brucei it is also found in the sub-telomeric variant surface glycoprotein (VSG) gene expression sites involved in antigenic variation [3][4][5]. By periodically switching its VSG coat the trypanosome is able to evade the host immune system in chronic infections.…”

“…In the DNA of kinetoplastid flagellates, a fraction of thymine is replaced by the modified base β-D-glucosyl-hydroxymethyluracil, called J [1][2][3]. This hyper-modified base consists of the attachment of a bulky glucose moiety to the thymine base such that it extends into the major groove of the DNA helix.…”