Biomedical subjects
Publications and source records attributed to E L McKay.
The DNA methylase activity present in embryos and cultured cells of Xenopus laevis resembles DNA methylase from mammalian tissues. Little or no activity is found in mature germinal vesicles, though nuclear activity rises rapidly after fertilization. This rise may result in part from a relocation of cytoplasmic enzyme.
Cells of the mosquito Aedes albopictus have 5.6 pg DNA/cell. This DNA is 58 mol% (A + T) and has about 0.03 mol% each of 5-methylcytosine and 6-methylaminopurine. The DNA is largely degraded by the restriction enzyme HpaII showing the virtual absence of the sequence CMeCGG. Small amounts of enzymic activity recovered largely in non-nuclear fractions transfer methyl groups from S-adenosylmethionine to cytosine and adenine.
An improved method of purification of DNA methylase from Krebs II ascites cells is reported. The enzyme sediments at 8.3 S on glycerol-gradients and a major band on SDS polyacrylamide gel electrophoresis has a molecular weight of 184 000. Aggregation occurs at low salt and this may interfere with enzymic activity. The preferred double stranded DNA substrate is that rendered partially unmethylated by an in vitro repair mechanism or by isolation from methionine starved cells. Methylation of native partially methylated DNA is favoured under conditions of low salt and high temperature; conditions which encourage 'breathing' of the DNA. Methylation of native, unmethylated DNA also involves breathing but results in formation of a salt resistant tight binding complex between the enzyme and the DNA.
The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.
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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 proportion of cytosines methylated in the DNA of nucleosome oligomers and of core particles appears indistinguishable from that of total nuclear DNA from CHO cells. However the DNA in nucleoprotein which is initially released from nuclei by treatment with very low levels of micrococcal nuclease and the first 10% of material rendered acid soluble by treatment of nuclei with DNase I are enriched 2 fold in their content of 5 methylcytosine. (Cessation of hydrolysis by nuclease occurs concomitantly with precipitation of nucleosomal core particles).