BIOSYNTHESIS AND FUNCTION OF THE MODIFIED DNA-BASE BETA-D-GLUCOSYL-HYDROXYMETHYLURACIL IN TRYPANOSOMA-BRUCEI

beta-D-Glucosyl-hydroxymethyluracil, also called J, is a modified DNA base conserved among kinetoplastid flagellates. In Trypanosoma brucei, the majority of J is present in repetitive DNA but the partial replac ement of thymine by J also correlates with transcriptional repression of the variant surface glycoprotein (VSG) genes in the telomeric VSG g ene expression sites, To gain a better understanding of the function o f J, we studied its biosynthesis in T. brucei and found that it is mad e in two steps, In the first step, thymine in DNA is converted into hy droxymethyluracil by an enzyme that recognizes specific DNA sequences and/or structures. In the second step, hydroxymethyluracil is glucosyl ated by an enzyme that shows no obvious sequence specificity. We ident ified analogs of thymidine that affect the J content of the T. brucei genome upon incorporation into DNA, These analogs were used to study t he function of J in the control of VSG gene expression sites. We found that incorporation of bromodeoxyuridine resulted in a 12-fold decreas e in J content and caused a partial derepression of silent VSG gene ex pression site promoters, suggesting that J might strengthen transcript ional repression, Incorporation of hydroxymethyldeoxyuridine, resultin g in a 15-fold increase in the J content, caused a reduction in the oc currence of chromosome breakage events sometimes associated with trans criptional switching between VSG gene expression sites in vitro. We sp eculate that these effects are mediated by the packaging of J-containi ng DNA into a condensed chromatin structure.