RECOGNITION OF URACIL RESIDUES IN DNA BY URACIL DNA GLYCOSYLASE FROM HUMAN PLACENTA

The dependence of the affinity to uracil DNA glycosylase and length wa s studied for homooligonucleotides d(pT)(n) and d(pA)(n) and a duplex d(pT)(n)d(pA)(n). The affinity of duplexes containing a dU residue was also estimated. Simple algorithms for evaluating the affinity of olig onucleotides to glycosylase were developed. It was shown that d(pT)(n) and d(pA)(n) inhibit the reaction catalyzed by uracil DNA glycosylase competitively against [H-3]DNA. The minimal ligands for the enzyme pr oved to be dTMP (K-i = 45 mu M) and dAMP (K-i = 10 mu M). At n 10, add ition of one unit to d(pT)(n) and d(pA)(n) increased their affinity (f ) 1.28 and 1.36 times, respectively, according to the following progre ssion: K-i[d(pN)(n)] = K-i(dNMP).(f)(-g), where g is the number of mon onucleotide units in the ligand. The affinity of d(pT)(n)d(pA)(n) chan ged in a similar manner, but its K-i values were approximately three t imes lower than those for d(pA)(n) at any n. Introduction of a dU resi due into d(pT)(n) d(pA)(n) increased its affinity 10-20-fold. The data obtained indicate that uracil DNA glycosylase interacts with ten base pairs of the duplex, the contribution of the dU residue to the affini ty being 3-4 orders of magnitude lower than that of the other nine uni ts together.