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A rapid and effective method of testing of a repair enzyme, uracil-DNA-glycosylase, was proposed. As a substrate, a deoxyuridine-containing 5'-32P-labeled deoxyoligonucleotide covalently attached to a polystyrene support (Tenta Gel S-NH2) was used. The ammonia cleavage of the apyrimidine site formed in the enzymic reaction followed by the transition of the labeled oligonucleotide fragment from the solid phase into solution allowed the detection of the enzymic activity.

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 n > 10 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.

Levels of unscheduled DNA synthesis after UV-irradiation were investigated in addition to the activities of DNA-repair enzyme uracil-DNA glycosilase in cells of both diploid (XC-D) and tetraploid (XC-T) cell cultures. It was shown that repair levels were increasing directly and proportionally to the cell ploidy.

The data confirming the formation of dUMP residues in DNA to be a continuous process taking place in the living cells are reviewed. All living organisms produce specific enzymes repairing the lesion of this type. The possible ways for uracil incorporation into DNA are described. The main of them are as follows: cytosine deamination in DNA molecules and utilization of dUTP by DNA polymerases during replication. The spontaneous mutability, the decrease in chain length of the newly synthesized DNA and the increase in recombination frequencies are discussed as possible consequences of this phenomenon.

The activity of uracil-DNA glycosylase (UDG) was studied for livers of 13 mammalian species belonging to four orders. DNA contents were also measured in isolated hepatocytes. The enzymatic activity was shown to increase with the increase in the mean ploidy of liver parenchymal cells. The activity of UDG was 20 times as high when the mean liver cell ploidy of different mammalian species doubled. A reverse dependence between the UDG activity and species life spans is also revealed.

Uracil-DNA-glycosylase was isolated from human placenta and purified 2100-fold. The apparent Km value for non-methylated DNA substrate of the enzyme is 3.10(-7) M. However, Km for uracil-DNA-glycosylase was 3 times as low when methylated DNA was used as a substrate. It was shown that the initial rate of uracil excision was greater for the non-methylated than for the hypermethylated DNA. The experimental results indicate that the postreplicative methylation of DNA can interfere with uracil excision.

Survival of phage lambda cI857 inactivated by bisulfite (pH 5.6, 37 degrees C) is higher (the dose modification factor approx. 1.2) and frequency of bisulfite-induced c-mutations 2-4-fold lower on the lawn of the wild-type strain ung+, as compared to ung-1 mutant deficient in uracil-DNA glycosylase. Irradiation of host cells by a moderate UV dose inducing SOS repair system enhances the frequency of bisulfite-induced c-mutations 2-3-fold in the wild-type (ung+) host, but not in the ung-1 mutant. It is suggested that W-mutagenesis in bisulfite-treated lambda phage in the ung+ cells is due to SOS repair of apyrimidinic sites which are produced during excision of uracil residues, the products of cytosine deamination.

A study was made of the lethal and mutagenic effects on extracellular phage gamma of 5-3H-cytosine incorporated into DNA. The efficiencies of inactivation by incorporated 3H were equal for 5-3H-cytosine and [3H-methyl]-thymidine, but the yield of c-mutations for the former was 14 times higher. The lethal and mutagenic effects of incorporated 5-3H-cytosine did not depend on ung mutation of host cells which caused a deficiency in uracil-DNA-glycosylase. The mutagenic effect was not enhanced when SOS-repair system was induced by UV-radiation. The mutagenic effect of 5-3H-cytosine was associated with the modified mispairing bases but not with uracil residues.

Uracil-DNA-glycosylase which releases uracil residues from DNA has been purified from rat liver more than 300-fold. The enzyme has a molecular weight of about 28,0000, Km = 1.7 . 10(-9) M. The content of uracil-DNA-glycosylase in five different tissues of the rat is correlated with the rate of DNA synthesis in the tissues. The enzyme activity in rat liver increases more than 3 times after partial hepatectomy, showing a peak 28-32 hrs following the surgery. The results obtained suggest that uracil-DNA-glycosylase from mammalian cells releases uracil residues from DNA at time intervals close to those of replication.

Immune antiserum to uracil-DNA glycosylase was obtained by immunizing rabbits with an enzyme isolated from the rat liver. Antiserum was found to suppress the activity of uracil-DNA glycosylase not only in the extracts of rat liver, but also in the extracts of brain, cardiac muscle, kidney, spleen, thymus of rats, and in those of human placenta too. This enables us to make a conclusion about the similarity in antigenic properties of the enzyme in cells of various types of differentiation. Indirect immunofluorescent test shows a slight staining of the periphery of the nucleus in normal liver hepatocytes and the intensive staining of the inner part of the nucleus in hepatocytes of regenerating liver. Therefore it is concluded that the enzymatic activity increases as cells proliferate. This may be the result of the appearance of uracil in DNA during replication.

Uracil-DNA glycosylase (UDG) purified by various procedures from the human placenta was used to obtain immune antisera with specific antibodies, the antibodies being affinity-purified on UDG-sepharose. Two immunoreactive polypeptides were found in crude extracts of the human placenta with the help of the antibodies. Their apparent molecular masses were about 37,000 and 34,000 dalton. Only the former polypeptide was found in crude extracts of the human embryonal heart, liver and in HeLa cells. The indirect immunofluorescent staining shows both slight and intensive fluorescence of HeLa cell nuclei. The similarity of antigenic properties of the human and rat UDG was confirmed.