Richard Y.-H. Wang

DNA cytosine methylation and heat-induced deamination

The heat-induced conversion of 5-methylcytosine (m5C) residues to thymine residues and of cytosine to uracil residues in single-stranded DNA was studied. The calculated rates for deamination at 37°C and pH 7.4 were ∼9.5×10−10 and 2.1×10−10 sec−1, respectively. N4-Methyldeoxycytidine, which is in the DNA of certain thermophilic bacteria, was more heat-resistant than was deoxycytidine and much more than was 5-methyldeoxycytidine. Thermophilic bacteria which contain N4-methylcytosine rather than m5C in their genomes may thereby largely avoid heat-induced mutation due to deamination, which is incurred by the many organisms that contain m5C in their DNA.

Heat- and alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA.

5-methylcytosine residues in DNA underwent deamination at high temperatures. Furthermore, their rate of deamination at neutral or alkaline pH was greater than that of cytosine residues in DNA. As sources of [14C]-5-methylcytosine-containing DNA, we used bacteriophage XP-12 DNA, in which 5-methylcytosine residues completely replace C residues, and calf thymus DNA experimentally substituted with [14C] 5-methylcytosine residues. Upon incubation at 95 degrees C in a physiological buffer or at 60 degrees C in 1 M NaOH, the respective rates of deamination of 5-methylcytosine residues were about 3- and 1.5-times those on cytosine residues. Under the same conditions, the free 5-methyldeoxycytidine was converted to thymidine more rapidly than deoxycytidine was converted to deoxyuridine. The reactions at physiological pH and elevated temperature suggest that deamination of 5-methylcytosine residues may yield a significant portion of spontaneous mutations in vivo, especially in view of the lack of thymine-specific mismatch repair systems with specificity and efficiency comparable to that of uracil excision repair systems.

Human DNA sequences exhibiting gamete-specific hypomethylation.

Three human DNA sequences have been cloned from DNA regions which are strikingly undermethylated in sperm, highly methylated in adult somatic tissues, and methylated to an intermediate extent in tissues of extraembryonic origin. It is proposed that some such DNA sequences may function specifically early in embryogenesis or during gametogenesis. They may be subsequently extensively methylated in the embryonic cell lineage and methylated to a lesser extent in extraembryonic tissues in order to allow embryogenesis to proceed.

A bacteriophage-induced 5-methyldeoxycytidine 5′-monophosphate kinase

Bacteriophage XP-12-infected Xanthomonas oryzae have been found to be a source of a kinase preparation which converts m5dCMP to m5dCDP and then to m5dCTP using ATP as the phosphate donor. Optimal formation of the triphosphate required the presence of creatine phosphate and creatine kinase. In the presence of dGTP, dTTP and dATP, Escherichia coli DNA polymerase I and T4 DNA polymerase catalyzed the incorporation of m5dCTP into DNA just as efficiently as that of dCTP. Neither dTMP nor dCMP served as substrate for the m5dCMP monophosphate kinase. Analogous preparations from uninfected X. oryzae were unable to phosphorylate m5dCMP.

Enzymatic conversion of deoxycytidine 5′-monophosphate to 5-methyldeoxycytidine 5′-triphosphate

Abstract An enzymatic method has been developed to synthesize 5-methyldeoxycytidine triphosphate (m5dCTP) starting with dCMP and formaldehyde as substrates. Methylation is catalyzed by a dCMP methyltransferase in the presence of tetrahydrofolic acid. The resulting m5dCMP is converted to m5dCTP by a kinase preparation and an ATP-regenerating system. The methyltransferase and kinase preparations were isolated from extracts of bacteriophage XP-12-infected Xanthomonas oryzae by a single-step chromatography on DEAE-cellulose. About 10% of the input radioactivity from 14C- or 3H-labeled formaldehyde was recovered in m5dCTP under our reaction conditions. Using this enzymatically synthesized m5dCTP, up to 60% of the cytosine residues in DNA was replaced by 5-methylcytosine residues in a nick translation reaction catalyzed by Escherichia coli DNA polymerase I.