PubMed · 15786803
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Trevor S Brown, Philip C Bevilacqua. 2005. Method for assigning double-stranded RNA structures.. https://doi.org/10.2144/05383bm04
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The Escherichia coli T4 bacteriophage uses two glycosyltransferases to glucosylate and thus protect its DNA: the retaining alpha-glucosyltransferase (AGT) and the inverting beta-glucosyltransferase (BGT). They glucosylate 5-hydroxymethyl cytosine (5-HMC) bases of duplex DNA using UDP-glucose as the sugar donor to form an alpha-glucosidic linkage and a beta-glucosidic linkage, respectively. Five structures of AGT have been determined: a binary complex with the UDP product and four ternary complexes with UDP or UDP-glucose and oligonucleotides containing an A:G, HMU:G (hydroxymethyl uracyl) or AP:G (apurinic/apyrimidinic) mismatch at the target base-pair. AGT adopts the GT-B fold, one of the two folds known for GTs. However, while the sugar donor binding mode is classical for a GT-B enzyme, the sugar acceptor binding mode is unexpected and breaks the established consensus: AGT is the first GT-B enzyme that predominantly binds both the sugar donor and acceptor to the C-terminal domain. Its active site pocket is highly similar to four retaining GT-B glycosyltransferases (trehalose-6-phosphate synthase, glycogen synthase, glycogen and maltodextrin phosphorylases) strongly suggesting a common evolutionary origin and catalytic mechanism for these enzymes. Structure-guided mutagenesis and kinetic analysis do not permit identification of a nucleophile residue responsible for a glycosyl-enzyme intermediate for the classical double displacement mechanism. Interestingly, the DNA structures reveal partially flipped-out bases. They provide evidence for a passive role of AGT in the base-flipping mechanism and for its specific recognition of the acceptor base.
Fluorescence microscopy was used to study the folding transition of giant DNAs, T4 DNA (ca. 166 kbp), and lambda DNA (ca. 48 kbp), which proceeds through intermediates with intramolecular segregation induced by pteridine-polyamine conjugates, i.e., 2-amino-6,7-dimethyl-4-(4,9,13-triazatridecylamino)pteridine and -4-(3-(aminopropyl)amino)pteridine. According to the results of DNA denaturation, UV and fluorescent spectroscopy, and transmission electron microscopic observations, it became clear that DNA folding induced by the polyamine derivative is not a continuous shrinking process but a combination of discontinuous processes.
Pteridine-polyamine conjugates, such as 2-polyamine (1,3-diaminopropane and spermine) substituted 6,7-dimethyl-3H-pteridine-4-one, induce a folding transition of a giant DNA molecule more effectively than the corresponding polyamines. However, since neither a DNA high-temperature shift of denaturation (Tm) curve nor distortion of the UV/fluorescence spectra is observed in a mixture of these compounds with DNA, they do not interact with the DNA duplex strongly.