Purification and properties of a novel DNA methyltransferase from cultured rice cells.

DNA methylttransferase activity has been observed in a total homogenate of rice cells grown in suspension culture using either native plant DNA or, under the conditions used, the more responsive hemimethylated poly (dI-MedC)*poly(dI-dC). Using the latter substrate we have purified an enzyme fraction 380-fold by salt extraction of chromatin, DEAE cellulose and phosphocellulose. This purified fraction showed enzyme activity only, with poly (d1-MedC)*poly(dI-dC) thus suggesting the occurrence in plants of a DNA methyltransferase specific for hemimethylated DNA. A Mr value of 54000 was calculated on the basis of the sedimentation coefficent which was determined by sucrose density gradient centrifugation. Apparent Km values for poly(d1- MedC)*poly(dI-dC) and S-adenosyl-L-methionin e were foundto be 17 µg/ml and 2.6 µM, respectively

Daucus carota cells contain specific DNA methyltransferase inhibitors that interfere with somatic embryogenesis

ABSTRACT Results reported in this paper show that carrot cells contain a thermostable inhibiting activity of cytosine-5-DNA methyltransferase that upon filtration chromatography can be resolved in three major peaks. An inhibiting activity was found in all plant species tested, though at a concentration lower than in carrot. These inhibiting activities differ in size, sensitivity to various hydrolytic treatments, specificity for DNA METases of eukaryotic and bacterial origin and kinetic of inhibition. Results of chemical analyses indicate that the inhibitors are different from lipidic inhibitors described in Escherichia coli and Streptomyces sp. and, for their sensitivity to proteinase K appear to have a proteinaceous nature. The addition of these inhibitors (Sephadex G 25 peak II and peak III) to actively growing suspension rice cells reduced the rate of in vivo DNA methylation without interfering with DNA synthesis. Peak II also induced a general demethylation effect in carrot cell suspension even if weaker than that caused by 5-azacytidine. Interestingly, inhibitors suppressed carrot embryogenesis but did not prevent undifferentiated cell proliferation of suspension cultures

DNA unwinding and inhibition of T4 DNA ligase by anthracyclines

The ability to alter DNA tertiary structure of ten anthracycline derivatives whose antitumor potency is known was studied by an assay that makes use of nicked circular DNA and bacteriophage T4 DNA ligase. This assay allows the detection of tertiary structure alterations caused by DNA binding of both intercalating and non-intercalating drugs. The determination of these events can be obtained at different temperatures in the range of activity of DNA ligase. The results indicate that anthracyclines alter the DNA tertiary structure but this property does not correlate with their cytotoxic or antitumor activities. An additional interesting finding was that several anthracyclines inhibit T4 DNA ligase. The inhibition can be complete and is a cubic function of drug concentration. The inhibition of DNA ligase does not correlate with the ability of anthracyclines to alter the tertiary structure of DNA but is dependent from the presence of an amino group on the sugar ring

Levels of DNA polymerase-alpha and beta in normal and xeroderma pigmentosum fibroblasts.

We have determined the levels of DNA-polymerases-alpha and-beta in fibroblasts obtained from normal subjects and from patients with Xeroderma Pigmentosum (XP) belonging to three different complementation groups and to the variant form. The assays have been performed in crude extracts and after fractionation on sucrose gradients. The levels of alpha and beta-polymerases in the different cases of XP were found to lie within the same range as the control values, and no correlation was found with the severity of the symptoms. The sedimentation coefficients of the two polymerases from all the pathological lines were identical to those of the normal fibroblasts