Modified substrates as probes for studying uracil-DNA glycosylase.
In order to study the mechanism of action of uracil-DNA glycosylase (UDG) from human placenta, single-stranded (ss) and double-stranded (ds) oligodeoxyribonucleotides (oligos), containing deoxyuridine (dU) and a wide variety of their analogs were used. It was shown that UDG has a twofold preference for ss oligos over ds oligos and a twofold preference for intermolecular duplexes over similar hairpin-like duplexes. The replacement of dU with 1-(beta-D-2'-deoxy-threo-pentofuranosil)uracil (xU) or 1-(beta-D-3'-deoxy-threo-pentofuranosil)uracil (tU), which results in a change in sugar hydroxyl configuration, has no influence on UDG binding to such substrates, but inhibits uracil removal. A oligo containing 2'-deoxy-2'-fluorouridine (flU), with a 3'-endo conformation of modified sugar is recognized by UDG 100-200-fold less efficiently than the natural ones. F or Br atoms or a methyl group were introduced at position 5 of a dU residue in an oligo. It was shown that the nature of a substituent at this position is essential for UDG function.
Kubareva
E A
EA
A.N. Belozersky Institute of Physical and Chemical Biology, Moscow State University, Russia.
Volkov
E M
EM
Vinogradova
N L
NL
Kanevsky
I A
IA
Oretskaya
T S
TS
Kuznetsova
S A
SA
Brevnov
M G
MG
Gromova
E S
ES
Nevinsky
G A
GA
Shabarova
Z A
ZA
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D045647:DNA Glycosylases; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011485:Protein Binding; D013379:Substrate Specificity; D051981:Uracil-DNA Glycosidase
D009838:Oligodeoxyribonucleotides; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase