DNA methylase in mouse cell nuclei

Qureshi, Mumtaz Ali (1983) DNA methylase in mouse cell nuclei. PhD thesis, University of Glasgow.

Abstract

Extraction of isolated nuclei from Krebs II mouse ascites cells (or mouse L-929 cells) with low salt (0.2M-0.4M) is sufficient to solubilise the major proportion of DNA methylase. The properties of this enzyme were such as to suggest that it could perform a role in maintenance of DNA methylation pattern or catalyse the de novo methylation of unmethylated DNAs. A brief search for soluble nuclear factors that might modify the activity of this enzyme was carried out but no specific factors that would meaningfully modulate the activity of this enzyme were obvious. On the other hand a search for further DNA methylase activity in these nuclei revealed activity which remained bound to nuclear structures even after extraction with 2M NaC1. The occurrence of this enzyme activity in such nuclear structures which resist to solubilise with 2M NaC1 and the results from alkaline CsC1 density gradient analysis are suggestive that this enzyme activity is a part of replication complex (see Pardoll et al., 1980) and is possibly involved in maintenance of the DNA methylation pattern after DNA replication. The properties of bound DNA methylase have been compared to those of DNA methylase already solubilised at low salt concentration. Response to NaC1, pH and DNA substrates as well as sedimentation characteristic, indicate that bound DNA methylase is quite similar to soluble DNA methylase. The inhibition of DNA synthesis with hydroxyurea released bound DNA methylase activity into the soluble form, suggests that occurrence in the bound form is dependent on DNA replication. In cells treated with 5-azadeoxycytidine, more than 80% of the DNA methylase was lost from the soluble form and further experiments support the notion that it may be bound to DNA nuclear matrix in an inactive form. The solubilisation of bound DNA methylase has been achieved by using extremely large amount of micrococcal nuclease. Since this procedure was quite expensive an alternative method, the "abstraction" of DNA methylase from the bound form with denatured E. coli DNA was employed. However, despite this solubilisation the methylase remained bound to the 'abstracting' DNA. By using 30% ammonium sulpahte, the solubilisation of bound DNA methylase from nuclear components, with high yield, has been carried out. Such study revealed that whereas 2M NaC1 failed to dissociate enzyme from nuclear structure, the ammonium sulphate has proved to be an efficient reagent. In view of the results obtained after the treatment of cells with 5-azadeoxycytidine and with DNA synthesis inhibitors, a model showing the biological relationship between these two forms of DNA methylases has been presented.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: R H Burdon
• Keywords: Biochemistry
• Date of Award: 1983
• Depositing User: Enlighten Team
• Unique ID: glathesis:1983-73423
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jun 2019 08:56
• Last Modified: 14 Jun 2019 08:56
• URI: https://theses.gla.ac.uk/id/eprint/73423