Unusual Modification of Bacteriophage Mu DNA

Abstract

Bacteriophage Mu DNA was labeled after induction in the presence of [2- 3 H]adenine or [8- 3 H]adenine. Both Mu mom + · dam + DNA and Mu mom − · dam + DNA have similar N 6 -methyladenine (MeAde) contents, as well as similar frequencies of MeAde nearest neighbors. Both DNAs are sensitive to in vitro cleavage by R· Dpn I but resistant to cleavage by R· Dpn II. These results indicate that the mom + protein does not alter the sequence specificity of the host dam + methylase to produce MeAde at new sites. However, we have discovered a new modified base, denoted A x , in Mu mom + · dam + DNA; approximately 15% of the adenine residues are modified to A x . Although the precise nature of the modification is not yet defined, analysis by electrophoresis and chromatography indicates that the N 6 -amino group is not the site of modification, and that the added moiety contains a free carboxyl group. A x is not present in Mu mom + · dam + or Mu mom − · dam + phage DNA or in cellular DNA from uninduced Mu mom + · dam + lysogens. These results suggest that expression of the dam + and mom + genes are required for the A x modification and that this modification is responsible for protecting Mu DNA against certain restriction nucleases. Mu mom + · dam − DNA and Mu mom − · dam − DNA contain a very low level of MeAde (ca. 1 MeAde per 5,000 adenine residues). Since the only nearest neighbor to MeAde appears to be cytosine, we suggest that the methylated sequence is 5′... C-A * -C... 3′ and that this methylation is mediated by the Eco K modification enzyme.