Effect of 5-azacytidine on E. coli cells with different DNA-methylases

A correlation was found between the bacteriocide effect of 5-aza-C and the amount of cytosine DNA-methylases in E. coli cells. 5-Aza-C-DNA induced partial or complete inhibition of bacterial DNA-methylases with different site specificity; cytosine DNA-methylases were inhibited by the DNA more effectively than adenine DNA-methylase Eco dam. The inhibitory influence of 5-aza-C-DNA on cytosine DNA-methylases was due to the formation of stable inactive complexes between the enzyme and the non-methylating cytosine analog in the recognition sites. Cytosine DNA-methylase Eco RII formed a relatively firm bond with 5-aza-C-DNA, which could be disrupted by 1 M KCl; this disruption restores the DNA-methylase activity and the inhibiting capacity of 5-aza-C-DNA. Thus, the binding of cytosine DNA-methylase to 5-aza-C in DNA is noncovalent; the inhibition of the enzyme by 5-aza-C-DNA is reversible.     

Bactericidal effect of 5-azacytidine on Escherichia coli carrying EcoRII restriction-modification enzymes

5-Azacytidine was found to be bactericidal to Escherichia coli carrying plasmids specifying EcoRII restriction-modification systems, but not to the same strains lacking these plasmids. Of other base analogs tested, only 5(beta-D-ribofuranosyl)isocytidine had similar, although weaker, effects. Plasmids that had lost the EcoRII restriction-modification system did not confer sensitivity to 5-azacytidine. Mutants defective in the restriction function remained sensitive to the toxic effects of the drug; however, a mutant defective in the modification function lost most of the sensitivity to 5-azacytidine. For the bactericidal effect to be seen, the cells had to be growing; cells in the stationary phase of growth were not killed by the drug. The drug inhibited the methylase enzyme, and an inhibitor of the enzyme could be detected in vitro in extracts of cells that had been treated with 5-azacytidine. This nalidixic acid inhibited its formation. Coumermycin but not nalidixic acid antagonized the bactericidal effect of the drug; however, coumermycin was more effective in preventing the inhibition of the methylase by 5-azacytidine than was nalidixic acid.     

Photochemistry and photobiology of 5-azacytidine: effect of repair on photostabilization of Escherichia coli.

The photochemical stability of the anomalous nucleic acid base 5-azacytidine (z5Cyd) on irradiation at 254 nm is by about one order of magnitude less than that of cytidine (Cyd). Contrary to the photochemical behaviour, incorporation of z5Cyd into the nucleic acids of E. coli strains SR 20 (uvr+ rec+), SR 74 (uvr+ rec-) and SR 22 (uvr- rec+) produced a higher resistance to UV light. Only the SR 73 (uvr- rec-) strain was shown to have an increased UV sensitivity. This latter finding is in accord with the photochemical properties of z5Cyd. The results led to the conclusion that excision and recombination repair processes contribute to the observable protective effect. The fact that inhibition of excission repair by caffeine or proflavine of the mutant uvr+ rec- changes protection into sensitization supports this idea.     

The effect of methionine and 5-azacytidine on fragile X expression.

The cellular mechanism for the expression of the fragile site at Xq28 is unknown. We tested the effect of 5-azacytidine and methionine on fragile X expression in lymphocytes and lymphoblastoid cells in an attempt to determine if DNA methylation was involved. We were unable to demonstrate a consistent dosage effect of methionine on fragile X expression. While 5-azacytidine was found to inhibit the fragile X in both males and females, it did so only at relatively high concentrations. We conclude that the role, if any, of DNA methylation in fragile X expression is likely to be secondary, the primary effect being due to thymidylate depletion.     

Involvement of E. coli dcm methylase in Tn3 transposition

The effects of DNA methyltransferases on Tn3 transposition were investigated. The E. coli dam (deoxyadenosine methylase) gene was found to have no effect on Tn3 transposition. In contrast, Tn3 was found to transpose more frequently in dcm+ (deoxycytosine methylase) cells than in dcm- mutants. When the EcoRII methylase gene was introduced into dcm- cells (E. coli strain GM208), the frequency of Tn3 transposition in GM208 was dramatically increased. The EcoRII methylase recognizes and methylates the same sequence as does the dcm methylase. These results suggest that deoxycytosine methylase modified DNA may be a preferred target for Tn3 transposition. Experiments were also performed to determine whether the Tn3 transposase was involved in DNA modification. Plasmid DNA isolated from dcm- E. coli containing the Tn3 transposase gene was susceptible to ApyI digestion but resistant to EcoRI digestion, suggesting that Tn3 transposase modified the dcm recognition sequence. In addition, restriction enzymes TaqI, AvaII, BglI and HpaII did not digest this DNA completely, suggesting that the recognition sequences of TaqI, AvaII, BglI and HpaII were modified by Tn3 transposase to a certain degree. The type(s), the extent and mechanism(s) of this modification remain to be investigated.     

Enzymatic synthesis of 5-azacytidine 5'-triphosphate from 5-azacytidine.

5-Azacytidine 5′-monophosphate (5-aza-CMP) was synthesized enzymatically from 5-azacytidine (5-aza-C) in a reaction catalyzed by uridine-cytidine kinase. In a second step, 5-azacytidine 5′-triphosphate (5-aza-CTP) was synthesized enzymatically from 5-aza-CMP using CMP kinase and nucleoside diphosphokinase. Due to the chemical instability of the triazide ring of 5-azacytosine at neutral and alkaline pH, the enzymatic synthesis and purification of the nucleotides by ion exchange chromatography were performed at acid pH. The enzymatically synthesized 5-aza-CTP had an ultraviolet absorbance spectrum at pH 5.5 similar to the spectrum of 5-aza-C. In the DNA-dependent RNA polymerase reaction, 5-aza-CTP inhibited the incorporation of [³H]CTP, but [³H]UTP, into RNA.     

Effect of interaction between 5-azacytidine and DNA (cytosine-5) methyltransferase on C-to-G and C-to-T mutations in Escherichia coli.

The purpose of this study was to determine the effect of the Dcm cytosine methyltransferase on 5-azacytidine (5-azaC) mutagenesis in Escherichia coli. We used a Lac reversion assay to measure C-to-G and C-to-T mutations at a single, methylatable cytosine in the lacZ gene, in the presence and absence of Dcm. C-to-G mutations are stimulated by 5-azaC but are largely independent of Dcm. In contrast, C-to-T mutations are not stimulated by 5-azaC in either wild type or dcm cells. However, in cells which contain Dcm but are defective in very short patch repair, the normally high frequency of spontaneous C-to-T mutations is decreased by the analog in a dose-dependent manner.     

The effect of 5-azacytidine on the root meristem ofVicia faba

The primary roots ofVicia faba seedlings were placed in a solution of 5-azacytidine and their further growth was observed after being replaced in running tap water. No inhibition of elongation occurred during the action of the 10^?5 M solution of 5-azacytidine for 24 hours, but during subsequent cultivation in water in the absence of inhibitor, further growth was blocked. This inhibition could be overcome by cytidine, uridine, sodium azide, 5-azidomethyluracil and simultaneously with the 5-azacytidine solution. Inhibition was accompanied by a high incidence of chromosome stickiness and to a less extent by an incidence of chromosome aberrations. The occurrence of stickiness and chromosome aberrations was prevented by adding excess cytidine to the 5-azacytidine solution.     

The sequence specificity of a mammalian DNA methylase.

The sequence specificity of an extensively purified DNA methylase preparation from Krebs II mouse ascites cells has been examined. The enzyme appears to be highly sequence dependent. Moreover the sequence distribution of cytosine residues that are methylated, bears a very close resemblance to the sequence distribution of 5'-methyl cytosine found in vivo in a wide range of vertebrate cells and is consistent with methylation of cytosines in the sequence R-Yn-C-R.     

The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.

The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms.