Characterisation and Partial Purification of DNA Methylase From Pea (Pisum sativum)

Houlston, Claire Elizabeth (1993) Characterisation and Partial Purification of DNA Methylase From Pea (Pisum sativum). MSc(R) thesis, University of Glasgow.

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Prior to the start of this project, pea DNA methylase had been partially purified from pea shoot tips by gel filtration. Only one species of the enzyme was identified (Yesufu et al., 1991 ). In plants both CpG and CpNpG sites (where N is any base) are methylated and there is evidence that methylation of these sites might serve different functions (Hershkovitz et al., 1990 ). It is therefore possible that there might be more than one DNA methylase in plants. Three DNA methyltransferases are known to exist in E. coli (Marinus, 1987), and although only one methylase gene has been identified in mice, possible post-translational modification has resulted in three closely related forms of the enzyme (Bestor et al., 1988 ). Most methyltransferases recognize a single DNA sequence but in prokaryotes Flavobacterium okeanokoites displays different strand specificities while the Bacillus subtillis methyltransferase shows multiple sequence specificity (Gunthert and Trautner, 1984; Lx)oney et al., 1989; Sugisaki et al., 1989 ). One of the aims of this project was to refine the purification procedure so that separate enzyme species if any could be identified. Crude enzyme extract, E1, was shown to methylate synthetic DNA oligonucleotides representing CpG and CpNpG target sequences. A preference for hemimethylated over unmethylated substrate DNA was observed for both di- and trinucleotide target DNAs. DNA methylase was observed to have the highest affinity for hemimethylated CpNpG target DNA following competition studies and ligation of CpNpG DNA. The DNA-enzyme complex formed with this CpNpG substrate was observed to be salt resistant while DNA-enzyme complexes formed with the other substrates dissociated after varying intervals. This suggests that the enzyme forms a stable complex with hemimethylated trinucleotide DNA and is consistent with the idea that once bound to this substrate DNA it remains bound as suggested by competition studies and ligation of target DNA. Partial purification of pea DNA methylase(s) was achieved by affinity chromatography involving elution from heparin Sepharose and Q Sepharose. Enzymic activity was assayed with hemimethylated trinucleotide and unmethylated dinucleotide synthetic oligonucleotides. The peak of enzyme activity was observed in different fractions when assayed with the two substrates. SDS-PAGE revealed bands of high molecular weight in all active fractions which were found to react with an antibody to a component of the wheat methylase enzyme. The large number of bands was considered to be due to degradation of the enzyme through proteolysis. The addition of further protease inhibitors such as benzamidine led to the loss of enzymic activity. No distinct bands were visible on SDS-PAGE gels between fractions exhibiting different levels of methylation with different substrates. This is consistent with there being only one pea DNA methylase enzyme. The differences observed in relative activities with different substrates during purification might be due to different extents of proteolysis or to changes in conformation of the enzyme. Such changes might also explain the relative difference in activity with hemi- and unmethylated substrate DNA following heat treatment of extract E1 and purified samples and during early growth of pea shoots.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Additional Information: Adviser: R I P Adams
Keywords: Biochemistry
Date of Award: 1993
Depositing User: Enlighten Team
Unique ID: glathesis:1993-76330
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Dec 2019 09:15
Last Modified: 19 Dec 2019 09:15

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