Recognition of Base J in Duplex DNA by J-binding Protein

Abstract

␤-D-Glucosylhydroxymethyluracil, also called base J, is an unusual modified DNA base conserved among Kinetoplastida. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes. We have previously found a J-binding protein (JBP) in Trypanosoma, Leishmania, and Crithidia. We have now characterized the binding properties of recombinant JBP from Crithidia using synthetic J-DNA substrates that contain the glycosylated base in various DNA sequences. We find that JBP recognizes base J only when presented in double-stranded DNA but not in singlestranded DNA or in an RNA:DNA duplex. It also fails to interact with free glucose or free base J. JBP is unable to recognize nonmodified DNA or intermediates of J synthesis, suggesting that JBP is not directly involved in J biosynthesis. JBP binds J-DNA with high affinity (K d ‫؍‬ 40 -140 nM) but requires at least 5 bp flanking the glycosylated base for optimal binding. The nature of the flanking sequence affects binding because J in a telomeric sequence binds JBP with higher affinity than J in another sequence known to contain J in trypanosome DNA. We conclude that JBP is a structure-specific DNAbinding protein. The significance of these results in relation to the biological role and mechanism of action of J modification in kinetoplastids is discussed.In the DNA of kinetoplastid flagellates, a fraction of thymine is replaced by the modified base ␤-D-glucosylhydroxymethyluracil, called J (1-3). In all kinetoplastids, J is abundantly present in telomeric repeats (3). In the parasite Trypanosoma brucei, J is also found in the telomeric variant surface glycoprotein (VSG) 1 gene expression sites involved in antigenic variation (4, 5). The presence of J in 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 (3-9).It has been suggested that J is involved in long term transcriptional repression and that J could also suppress unwanted recombination between repetitive sequences in the genome (3,8,9). Whether this is true and whether J is the cause or the consequence of this silencing remain to be determined. However, consistent with both ideas, the protrusion of the sugar group of the major groove of DNA, at specific locations, could allow the recognition and binding of proteins that would mediate J function. These proteins could lead to gene silencing and/or suppression of DNA recombination, both of which are involved in the mechanism of antigenic variation. Our recent discovery of J-binding proteins (JBPs) in kinetoplastids that specifically bind J-containing DNA is compatible with this idea (10). By understanding how JBP specifically recognizes and binds the unique modified base in DNA could represent a first step in elucidating the function and mechanism of J action.In this report, we use recombinant JBP to study the interaction between JBP and J-DNA. We utilize DNA ...

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

“…Not only is JBP1 able to bind hmU-modified DNA (17), but detection of hmU formation by JBP1 in vitro peaks early during the reaction but then decreases over time. 5 Although we acknowledge that further work is needed to conclusively demonstrate JBP-stimulated conversion of hmU to fU and 5-carboxyluracil, the data thus far allow us to propose a model where JBP and JGT compete for hmU substrate in the trypanosome genome for conversion to base J or additional base analogs (fU and 5-carboxyluracil).…”

JBP1 and JBP2 Proteins Are Fe2+/2-Oxoglutarate-dependent Dioxygenases Regulating Hydroxylation of Thymidine Residues in Trypanosome DNA

“…The hydroxylation activity of JBP1 is specific for dsDNA, correlating with the specificity of J-DNA binding domain at the C terminus of JBP1 (34,35). Inactivation of the N-terminal TH domain has no effect on J-DNA binding (8).…”

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

“…The ability of JBP1 to bind J-DNA is essential for propagation [11] (Sabatini unpublished data). Also, JBP1 does not bind single-stranded J-DNA or the RNA/J-DNA duplex [13]. Thus, the high rate of polymerase I transcription could inhibit JBP1 stimulated J-propagation within the active expression site polycistronic transcription unit.…”

“…This apparent block in JBP1 stimulated J-propagation within the active 221 ES may be due to distance constraints or the effect of on-going transcription on the propagation mechanism. Based on the analysis of the JBP1/J-DNA interaction, we have previously proposed that the high rate of polymerase I transcription explains the exclusive lack of J in the active expression site [13]. The ability of JBP1 to bind J-DNA is essential for propagation [11] (Sabatini unpublished data).…”

“…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).…”