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Abstract
We reported here the use of T4 bacteriophage β-glucosyltransferase (T4 β-GT) for the facile synthesis of base J-containing oligodeoxyribonucleotides (ODNs). We found that the enzyme could catalyze the glucosylation of 5-hydroxymethyl-2-deoxyuridine (5hmU) in both single- and double-stranded ODNs, though the latter reaction occurred only when 5hmU was mispaired with a guanine. In addition, base J blocked moderately DNA replication, but it did not induce mutations during replication in human cells.
Highlights
Unicellular eukaryotic kinetoplastid flagellates, such as Trypanosoma and Leishmania species, contain a unique modified base, 5-(b-D-glucosylhydroxymethyl)uracil (a.k.a. base J), in their nuclear DNA (Figure 1) [1,2]
Base J is produced by oxidation of thymine to 5-hydroxymethyluracil and subsequent glucosylation of the latter modified nucleobase [3]
T4 bacteriophage b-glucosyltransferase (T4 b-GT) catalyzes the transfer of a glucose residue from UDP-Glc to 5-hydroxymethyl-29-deoxycytidine (5hmC) in double-stranded DNA, yielding 5-(b-glucosylhydroxymethyl)-29deoxycytidine (Glc-5hmC) (Figure 1) [26]. We reasoned that this enzyme may be employed for the glucosylation of 5hmU to yield base J in DNA
Summary
Unicellular eukaryotic kinetoplastid flagellates, such as Trypanosoma and Leishmania species, contain a unique modified base, 5-(b-D-glucosylhydroxymethyl)uracil (a.k.a. base J), in their nuclear DNA (Figure 1) [1,2]. The enzyme catalyzing the glucose transfer reaction involved in base J biosynthesis has yet been identified, though many experimental approaches including complementation in cell extracts and RNAi knockdown of candidate genes have been attempted [4] In this vein, it is worth noting that, through bioinformatic analysis of biochemical pathways for DNA modifications, Avarind et al [9] recently identified a putative glucosyltransferase with an operonic association to a JBP-related gene in several phage genomes. Understanding the biological function and characterizing the biophysical properties of base J at the molecular level necessitate the availability of oligodeoxyribonucleotides (ODNs) containing a site- inserted base J Along this line, base J-carrying ODNs have been previously synthesized using conventional phosphoramidite chemistry and automated solid-phase DNA synthesis [11,12,13]. In contrast to its strong blocking effects on DNA transcription, base J moderately impedes DNA replication in human cells and it does not induce mutations during this process
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