The phosphodiester bond 3' to a deoxyuridine residue is crucial for substrate binding for uracil DNA N-glycosylase.
Using the method of water-soluble carbodiimide-induced chemical ligation, four 27-member oligodeoxyribonucleotides containing a pyrophosphate internucleotide bond near or adjacent to a deoxyuridine residue were prepared. Escherichia coli uracil DNA N-glycosylase (UDG) activity was found to be sensitive to the presence of an internucleotide pyrophosphate bond in both single- and double-stranded DNA. The rate of uracil excision from single-stranded DNA containing a pyrophosphate bond adjacent to the uracil residue, either 3' or 5', was 0.01% and 0.1% of the rate of uracil removal from control DNA without a pyrophosphate bond, respectively. The rate of uracil excision from duplex DNA containing a pyrophosphate bond 3' or 5' to the uracil residue was also reduced, being 0.1% and 1% the rate of uracil removal from the corresponding duplex DNA control. Placing the pyrophosphate bond one nucleotide 5' or 3' away from the deoxyuridine in both single- and double-stranded oligodeoxyribonucleotides provided much better substrates for UDG. Kinetic measurements showed that the pyrophosphate bond placed adjacent to the deoxyuridine residue drastically reduced the affinity of UDG toward the modified DNA substrate, with the greatest effect occurring when the pyrophosphate bond was 3' adjacent to the deoxyuridine. The enzyme was able to excise a 3'-terminal uracil at the nicked site of a nicked duplex, DNA, provided that the terminal deoxyuridine was 3'-phosphorylated. The effect of the pyrophosphate bond on the substrate susceptibility of oligonucleotides containing deoxyuridine is discussed with respect to the mechanism of action of UDG.
Purmal
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AA
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington 05405, USA.
Wallace
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SS
Kow
Y W
YW
D001483:Base Sequence; D001665:Binding Sites; D045647:DNA Glycosylases; D003857:Deoxyuridine / Q000737:chemistry; D011756:Diphosphates / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013379:Substrate Specificity; D051981:Uracil-DNA Glycosidase
D011756:Diphosphates; D009838:Oligodeoxyribonucleotides; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D003857:Deoxyuridine