Unmethylated dam recognition sites in the Escherichia coli chromosome
Thesis DisciplineCellular and Molecular Biology
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
The dam (DNA adenine methylase) gene of Escherichia coli codes for an enzyme that converts adenine to N6-methyladenine at the sequence 5'GATC³’ shortly after DNA replication. dam mutants have pleiotropic phenotypes indicating that the enzyme and its target sequence may be involved in a number of cellular processes. In a normal cell about twenty GATC sites are stably unmethylated and many are partially methylated due to factors preventing methylation. A combinatorial analysis using restriction enzymes that cleave only unmethylated dam sequences and pulsed-field electrophoresis indicated the pattern and numbers of unmethylated sites were different in three common laboratory strains of E. coli K-12 (AB1157, W3110 & EMG2). Environmental factors such as nutrient levels, pH and stage of growth were found to influence unmethylated sites in strain AB1157. Changes in cell gene expression and chromosome organisation in response to these factors may be the cause for this observation. The global regulatory proteins Lrp and Crp also affected patterns of Dam methylation. Mutations at the lexA locus were found to have little influence on patterns of Dam methylation. Direct or indirect effects of these regulatory proteins binding DNA could be responsible for methylation exclusion at some unmethylated GATC loci. Sequencing of cloned AB1157 chromosomal unmethylated GATC sites revealed some unique loci where the protection mechanisms were not known and confirmed protection at a locus identified by another study. The distribution of unmethylated sites in the chromosome of AB1157 was found to follow no identifiable pattern.