J‐binding protein increases the level and retention of the unusual base J in trypanosome DNA

Abstract

SummaryThe nuclear DNA of Trypanosoma brucei and other kinetoplastid flagellates contains the unusual base b b b b -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.

Factors Affecting the Level and Localization of the Transferrin Receptor in Trypanosoma brucei

“…The HAC1A cell line also expresses the 221 expression site (31). In ⌬ESAG7::NEO trypanosomes, a neomycin gene cassette replaced ESAG7 of the active expression site (32).…”

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

“…JBP1 would then bind and propagate further J-synthesis. The JBP1 (−/−) trypanosome contains 20-fold less J than wild-type cells [10]. Our model of JBP2 function would predict that the basal level of J remaining in the JBP1 (−/−) (5%) is due to JBP2.…”

“…The search for proteins in bloodstream form nuclear extracts, able to bind J containing DNA, led to the identification of the first co-factor involved in J-biosynthesis: JBP1. JBP1 is a J-DNA specific binding protein involved in the maintenance and propagation of base J in the trypanosome genome [9][10][11]13]. JBP1 requires residual levels of J in the genome to bind and directly stimulate further synthesis along the DNA (propagation) as well as, presumably, on the opposing strand after replication (inheritance).…”

“…Therefore, we have proposed that JBP2 recognizes and binds certain regions of the chromosome, hydrolyses ATP and allows the Jsynthesis machinery to gain access to condensed chromatin by altering chromatin architecture [11]. JBP1 then binds to this basal level of localized J and stimulates additional synthesis by recruitment of the machinery to DNA [10,11].…”

“…While all attempts to isolate TH and GT activities have failed thus far, the identification of a base J binding protein (JBP1) in bloodstream-form T. brucei nuclear extracts led to the characterization of the first co-factor involved in J biosynthesis [9]. Analysis of the bloodstream form JBP1 (−/−) trypanosome, and ectopic expression in procyclic-form trypanosomes, has indicated that JBP1 represents a propagation and inheritance factor for J-biosynthesis [10,11]. JBP1 binds J-DNA and presumably activates (i.e.…”