The telomeric GGGTTA repeats of Trypanosoma brucei contain the hypermodified base J in both strands

Abstract

We have previously shown that nuclear DNA of bloodstream from Trypanosoma brucei contains a novel base beta-glucosyl-hydroxymethyluracil, called J. Base J is enriched in minichromosome fractions but not in the minichromosome internal repeats, suggesting the association of J with telomeric DNA. To test whether J is present in the long telomeric (GGGTTA)n repeat arrays, which are 2-26 kb in T.brucei, we have purified these arrays both by hybrid selection and by isolating 2-26 kb fragments from DNA digested with multiple restriction enzymes. We find that in purified telomeric repeats approximately 13% of T is replaced by J, compared to 0.8% in total DNA, and we estimate that approximately 50% of the total J is in these repeats. Highly purified complementary strands of the repeats were obtained by alkaline CsCl equilibrium centrifugation. In the (TAACCC)n strand 14% of T was replaced by J. In the (GGGTTA)n strand approximately 36% of the second T was replaced by J; the first T was not detectably replaced. Modified bases have not been found in telomeric repeats before. How the bulky base J affects telomere function and structure in bloodstream form trypanosomes remains to be determined.

Trypanosome Telomeres Are Protected by a Homologue of Mammalian TRF2

“…First, tbTRF is a duplex telomere DNAbinding factor. It is interesting that tbTRF has higher affinity for double-stranded TTAGGG repeats than for any permuta- (73,74), and two J-binding proteins have been identified (20,59). Thus, tbTRF may bind the J-containing repeats with lower affinity, which also appears to be the case for hTRF1 expressed in T. brucei (52).…”

Modification of Subtelomeric DNA

“…NlaIII sites are sensitive to adenine methylation in bacterial cells (22,25), but such modifications have not been observed in mammalian DNA. The thymidine modification beta-D-glucosylhydroxymethyluracil (base J) is present in repetitive sequences, primarily telomeric DNA, of kinetoplastid protozoans such as trypanosomes (10,11,29). It is involved in vesicular stomatitis virus gene silencing and produces resistance to enzymatic digestion.…”

“…The extremely repetitive nature of telomeres, the normal lack of recombination as a length maintenance mechanism, and the linkage disequilibrium including subtelomeric regions suggest the possibility of extensive base modifications at telomeres. Although unusual base modifications have been described at trypanosome telomeres (29), no such modifications have yet been reported in human telomeres. The best evidence to date suggesting the presence of extensive subtelomeric DNA modifications is the proposed existence of an approximately 2-to 4-kb region of subtelomeric DNA that is resistant to enzymatic digestion (18) and thus contributes to the apparent size of telomeres on gels.…”

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

“…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.…”