In vitro methylation of bacteriophage λ DNA by wild type ( dam +) and mutant ( damh) forms of the phage T2 DNA adenine methylase

Abstract

The wild-type ( dam + ) and mutant ( dam h) forms of the bacteriophage T2 DNA adenine methylase have been partially purified; these enzymes methylate the sequence, 5/t' … G-A-Py … 3′ (Hattman et al., 1978 a). However, in vitro methylation studies using phage λ DNA revealed the following: (1) T2 dam + and dam h enzymes differ in their ability to methylate λ DNA; under identical reaction conditions the T2 dam h enzyme methylated λ DNA to a higher level than did the dam + enzyme. However, the respective methylation sites are equally distributed on the l and r strands. (2) Methylation with T2 dam h, but not T2 dam + protected λ against P1 restriction. This was demonstrated by transfection of Escherichia coli (P1) spheroplasts and by cleavage with R· EcoP1. (3) T2 dam + and dam h were similarly capable of methylating G-A-T-C sequences on λ DNA; e.g. λ· dam 3 DNA (contains no N 6-methyladonine) methylated with either enzyme was made resistant to cleavage by R· DpnII. In contrast, only the T2 dam h modified DNA was resistant to further methylation by M· EcoP1 (which methylates the sequence 5′ … A-G-A-C-Py … 3′; Hattman et al., 1978 b). (4) λ· dam 3 DNA was partially methylated to the same level with T2 dam + or T2 dam h; the two enzymes produced different patterns of G-A-C versus G-A-T methylation. We propose that the T2 dam + enzyme methylates G-A-C sequences less efficiently than the T2 dam h methylase; this property does not entirely account for the large difference in methylation levels produced by the two enzymes.