The Detection of Extremely Rare DNA Modifications: Methylation in dam− and hsd−escherichia coli strains

Abstract

Abstract DNA methylation in Escherichia coli plays a role in many key cellular processes, including DNA replication, repair, restriction, and transcription. However, several mutant bacterial strains exist which are deficient in DNA methylase activities. Thus, it has been suggested that methylation produced by the dam (DNA adenine methylase) gene is required for the viability of E. coli and that dam- strains still produce low levels of methylation. Current experimental methods are not sensitive enough to detect a few potentially essential methylated sites per genome. Here we describe a method for the detection of N6-methyladenine at specific sites with a sensitivity of one site in more than 10 megabases. We show that methylation produced by both the dam and hsd (EcoK) genes is not required for the growth of E. coli and identify the site of EcoK modification. Minor adaptations of the technique should enable the identification of other rare DNA modifications.