Studies on DNA methylase from Ascites cells

Turnbull, John F (1976) Studies on DNA methylase from Ascites cells. PhD thesis, University of Glasgow.

Abstract

The DNA of higher eukaryotes contains small quantities of the minor base B-methylcytosine, formed by enzymatic transfer of methyl groups from S-adenosyl-L-methionine (SAM) to cytosine residues on polymeric DNA. The function of this methylated base is uncertain. In order to examine the mechanism and specificity of methylation, DNA methylase has been purified some 400-fold from Krebs II ascites tumour cells. This enzyme preparation has the following properties: (1) There is no evidence for more than one enzyme species. (2) The only product of methylation by the enzyme is 5-methylcytosine in DNA. Methylation is absolutely dependent on added substrate DNA. (3) The enzyme methylates DNA from a variety of animal, cultured cell and bacterial sources, but at widely differing rates. Higher rates of in vitro methylation are generally observed with DNAs from rapidly dividing cells. C4) Several experiments on the mechanism of action of the enzyme indicate that it operates by a series of repeated transient binding events at different sites in the DNA, rather than by an initial binding at a specific site follov/ed by a linear traverse of the DNA helix. (5) The enzyme can methylate both native and denatured calf thymus DNA; in E.coli DNA, however, only single-stranded regions are methylated even in a "native" DNA preparation. This could be caused by the enzyme requiring hemimethylated sites in double-stranded DNA, which are presumably absent from E. coli DNA. (6) Methylation of denatured DNA by the enzyme shows a strikingly greater resistance to inhibition by salt than does methylation of native DNA. The explanation suggested for this is that native and denatured DNA are methylated by different forms of the enzyme, perhaps by a dimer and a monomer respectively. This is based on the results of gel filtration and density gradient centrifugation, which show that under high salt conditions the enzyme exists as a smaller species than at low salt. Although not precisely determined, the approximate molecular weights of these species are consistent with the larger being a dimer of the smaller, which has a molecular weight of about 160,000.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Roger Adams
• Keywords: Biochemistry
• Date of Award: 1976
• Depositing User: Enlighten Team
• Unique ID: glathesis:1976-72346
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 24 May 2019 15:12
• Last Modified: 24 May 2019 15:12
• URI: https://theses.gla.ac.uk/id/eprint/72346