The telomeric GGGTTA repeats of Trypanosoma brucei contain the hypermodified base J in both strands.
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.
van Leeuwen
F
F
Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Wijsman
E R
ER
Kuyl-Yeheskiely
E
E
van der Marel
G A
GA
van Boom
J H
JH
Borst
P
P
D000818:Animals; D001483:Base Sequence; D016054:DNA, Protozoan / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D018503:Genome, Protozoan; D005960:Glucosides / Q000032:analysis*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012091:Repetitive Sequences, Nucleic Acid; D016615:Telomere / Q000737:chemistry / Q000235:genetics*; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000235:genetics*; D014498:Uracil / Q000031:analogs & derivatives* / Q000032:analysis
D016054:DNA, Protozoan; D004277:DNA, Single-Stranded; D005960:Glucosides; C084880:5-((glucopyranosyloxy)methyl)uracil; D014498:Uracil