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Summary The nuclear DNA of Trypanosoma brucei and other kinetoplastid flagellates contains the unusual base β-d-glucosyl-hydroxymethyluracil, called J, replacing part of the thymine in repetitive sequences. We have described a 100 kDa protein that specifically binds to J in duplex DNA. We have now disrupted the genes for this J-binding protein (JBP) in T. brucei . The disruption does not affect growth, gene expression or the stability of some repetitive DNA sequences. Unexpectedly, however, the JBP KO trypanosomes contain only about 5% of the wild-type level of J in their DNA. Excess J, randomly introduced into T. brucei DNA by growing the cells in the presence of the J precursor 5-hydroxymethyldeoxyuridine, is lost by simple dilution as the KO trypanosomes multiply, showing that JBP does not protect J against removal. In contrast, cells containing JBP lose excess J only sluggishly. We conclude that JBP is able to activate the thymine modification enzymes to introduce additional J in regions of DNA already containing a basal level of J. We propose that JBP is a novel DNA modification maintenance protein. [ABSTRACT FROM AUTHOR]

DNA from Kinetoplastida contains the unusual modified base β-D-glucosyl(hydroxymethyl)uracil, called J. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes in Trypanosoma brucei. We have now identified a protein in nuclear extracts of bloodstream stage T.brucei that binds specifically to J-containing duplex DNA. J-specific DNA binding was also observed with extracts from the kinetoplastids Crithidia fasciculata and Leishmania terentolae. We purified the 90 kDa C.fasciculata J-binding protein 50 000-fold and cloned the corresponding gene from C.fasciculata, T.brucei and L.tarentolae. Recombinant proteins expressed in Escherichia coli demonstrated J-specific DNA binding. The J-binding proteins show 43–63% identity and are unlike any known protein. The discovery of a J-binding protein suggests that J, like methylated cytosine in higher eukaryotes, functions via a protein intermediate. [ABSTRACT FROM AUTHOR]

Focuses on the deoxyribonucleic acid (DNA) base ...-D-glucosylhydroxymethyluracil known as `J' replacing ...0.5-1 percent of Thy in DNA of African trypanosomes. Methodology used to conduct the study; Development of sensitive asays to screen for J in a range of organisums; Discussion on the use of a sensitive anti-J immunobolt assay.