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The affinity of different ligands (d(pA)n, d(pT)n, d(pA)nxd(pT)n, dU-containing oligonucleotides) to the uracil-DNA-glycosylase (UDG) from human placenta have been investigated. All used oligodeoxynucleotides were shown to be competitive inhibitors of uracil-DNA glycosylase toward to [3H]-uracil-DNA substrate. Minimal ligand capable to bind to the template site of the enzyme was shown to be nucleoside monophosphate (Ki(dTMP) = 30 mM, Ki(dAMP) = 10 mM). Ligand affinity increases by the factor f 1.28 and 1.36 (respectively for d(pT)n and d(pA)n) per added monomer unit according to the progression Ki[d(pN)n] = Ki(dNMP).(f)-g, where g- number of mononucleotide bases of the d(pA)n, d(pT)n. Linear dependences of -lgKi vs n have inflection point at n = 10. At n10 ligand affinity remain constant. Affinity of the complexes d(pA)n.d(pT)n were observed to have the analogues dependencies, but Ki was 3 fold lower than for d(pA)n with corresponding length. The Ki of duplexes containing noncomplementary residues have been determined. Insertion of dU-residues or other noncomplementary base into one of the chains of duplexes d(pA)n.d(pT)n leads to increasing of the affinity about 10-20 fold: d(pT)4(pU)(pT)5 x d(pA)10 (Ki = 6.0 MKM), d(pT)10 x d(pA)4(pU)(pA)5 (Ki = 3.0 MKM), d(pA)7(pU)(pA)7 x d(pT)7(pC)(pT)7(4.0 MKM), d(pA)7(pU)(pA)7 x d(pT)7(pG)(pT)7 (6.0 MKM), d(pA)7(pU)(pA)7 x d(pT)15 (7.0 MKM). On the basis of the data obtained the conclusion that UDG interacts with 10 mononucleotide units of DNA was reached. The contribution of 9 nonmodified base pairs of DNA into recognition of substrate containing modified base by the enzyme is about 3-4 orders of magnitude higher than the contribution of the modified base. |
