After treatment of poly(dC) by the simple alkylating agent (3H)dimethylsulfate, 90 per cent of the radioactivity cochromatographied with 3-methylcytosine and 10 per cent with 5-methylcytosine which is the normally occurring methylated base. In order to study the influence of 3-methylcytosine on DNA replication, untreated and DMS-treated poly(dC) were used as templates for E. coli DNA polymerase I. The alkylation of poly(dC) inhibits DNA chain elongation, and does not induce any mispairing under high fidelity conditions. The alteration of DNA polymerase I fidelity by manganese ions allows some replication of 3-methylcytosine which mispairs with either dAMP or dTMP. Our results suggest that 3-methylcytosine could be responsible, at least partially, for the killing and the mutagenesis observed after cell treatment by alkylating agents.

Alkylating Agents↗

PubMedDate not supplied

Comparison at the molecular level of uracil-DNA glycosylases from different origins.

A nuclear and a cytoplasmic uracil-DNA glycosylase have been purified from epithelial cells derived from a rat hepatoma (H4 cells) cultured in vitro. They have different optimum pH, molecular weight, isoelectric points, activation energy, Km. Uracil acts as a non competitive inhibitor towards the nuclear enzyme while it is a competitive one for the cytoplasmic enzyme. Comparison of the properties of the two mammalian enzymes with those of the enzymes isolated from Escherichia coli and Micrococcus luteus shows that they all behave differently. The following criteria were studied: molecular weight, optimum pH, isoelectric point, inhibition by uracil analogs, modulation of their activity by polyamines or by intercalating drugs. The only common properties shared by these four enzymes are: an activity twice as high on single stranded DNA than on double stranded DNA and no requirement for divalent cation for maximal activity.

Animals↗

PubMedDate not supplied

Template properties of ultraviolet-irradiated poly(dC) replicated by E. coli DNA polymerase I: indication for a role of apyrimidinic-sites in UV-induced mutagenesis.

Ultraviolet irradiation alters the template properties of poly(dC) when replicated by Escherichia coli DNA polymerase I. These effects are due to base modifications. Some of them are identified as apurinic/apyrimidinic sites (AP-sites) by their sensitivity to AP-endonuclease B purified from Micrococcus luteus, and their template properties. The rate of formation of AP-sites in poly(dC) is estimated at 3 X 10(-7) site per nucleotide per J.m-2. Exposure of supercoiled or relaxed pBR322 DNA to UV light results also in the formation of sites sensitive to AP-endonuclease B. In this case, the rate of formation of AP-sites is the same in relaxed or supercoiled DNA: 0.3 X 10(-7) site per nucleotide per J.m-2. The apyrimidinic sites are generated through the processing of an ultraviolet induced primary lesion. We suggest that this lesion is cytosine hydrate by its rate of decay and preferential formation in single stranded DNA. Our results suggest that AP-sites might be a minor pathway leading to UV-induced mutagenesis.

Cytosine↗

PubMedDate not supplied

Chemical carcinogens and proto-oncogens activation.

The induction of proto-oncogens H-ras-1 by nitrosomethylurea (Sukumar et al. (1983), 306, 658-661) is discussed in term of lack of DNA-repair of lesions induced in DNA by this alkylating agent, particularly O6-methylguanine residues and apurinic/apyrimidinic sites.

Alkylating Agents↗

PubMedDate not supplied

Reaction kinetics for nitrosation of cysteine and glutathione in aerobic nitric oxide solutions at neutral pH. Insights into the fate and physiological effects of intermediates generated in the NO/O2 reaction.

The critical regulatory function of nitric oxide (NO) in many physiologic processes is well established. However, in an aerobic aqueous environment NO is known to generate one or more reactive and potentially toxic nitrogen oxide (NOx) metabolites. This has led to the speculation that mechanisms must exist in vivo by which these reactive intermediates are detoxified, although the nature of these mechanisms has yet to be elucidated. This report demonstrates that among the primary bioorganic products of the reaction of cellular constituents with the intermediates of the NO/O2 reaction are S-nitrosothiol (S-NO) adducts. Anaerobic solutions of NO are not capable of nitrosating cysteine or glutathione, while S-NO adducts of these amino acids are readily formed in the presence of O2 and NO. Investigation of the kinetics for the formation of these S-NO adducts has revealed a rate equation of d[RSNO]/dt = kSNO[NO]2[O2], where kSNO = (6 +/- 2) x 10(6) M-2S-1, a value identical to that for the formation of reactive intermediates in the autoxidation of NO. Competition studies performed with a variety of amino acids, glutathione, and azide have shown that cysteine residues have an affinity for the NOx species that is 3 orders of magnitude greater than that of the nonsulfhydryl amino acids, and > 10(6) times greater than that of the exocyclic amino groups of DNA bases. The dipeptide alanyltyrosine reacts with the intermediates of the NO/O2 reaction with an affinity 150 times less than that of the sulfhydryl-containing compounds. Furthermore, Chinese hamster V79 lung fibroblasts depleted of glutathione display enhanced cytotoxicity on exposure to NO.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Ring-opened alkylated guanine is not repaired in Z-DNA.

Since the discovery of Z-DNA by X-ray analysis of the alternated hexanucleotide d(C-G)3 crystals, numerous studies have shown that fragments of natural DNAs can adopt the Z conformation, topological constraints being a major factor stabilizing this conformation. Immunochemical assays using antibodies to Z-DNA provide strong evidence for the presence of Z fragments in chromosomes. The biological role of Z-DNA is not yet known, but it might be involved in gene regulation. Proteins which bind specifically to Z-DNA have been isolated and interactions between Z-DNA and several cellular proteins have been studied. The ability of DNA repair enzymes to maintain the genome's integrity is of major importance to the cell. On alkylation of DNA by chemical carcinogens such as dimethyl sulphate, methyl methanesulphonate, methylnitrosourea or methylnitrosoguanidine, the main target is the N7 of the guanosine residue, yielding 7-methylguanine (mG). In alkaline conditions, the imidazole ring of mG opens up, yielding the ring-opened form 2,6-diamino-4-oxo-5-methylformamidopyrimidine (rom7G); this lesion is a block to DNA replication. It occurs in vivo and is enzymatically removed by the DNA glycosylase. Here we report that the lesion is not excised when present in DNA in the left-handed Z conformation.

DNA Repair↗

PubMedDate not supplied

The quantitative distribution of certain enzymes along the small intestine of the rat and its correlation with the villous area and the Paneth cells.

The distribution of lysozyme, alkaline phosphatase, aminopeptidase, maltase and amylase was studied throughout the small intestine of the adult rat. Lysozyme activity increases along the length of the small intestine and the behaviour of this enzyme slightly differs from the mucosal enzymes reported in this investigation. A positive correlation is found between the percentage of crypts with granulated Paneth cells and the lysozyme activity. This corroborates with the secretory origin of this enzyme from these intestinal cells.

Alkaline Phosphatase↗