Dam methyltransferase sites located within the loop region of the oligopurine-oligopyrimidine sequences capable of forming H-DNA are undermethylatedin vivo

Several derivatives of pUC18 plasmid were constructed that contained oligopurine-oligopyrimidine (pur-pyr) motifs surrounded by Dam methylation sites. Inserts of two of the molecules (pPP1 and pPP2) were able to adopt the triple-stranded conformation in vitro and show in vivo a remarkable undermethylation of specific sites when grown in JM105 dam+ strain. Mapping experiments revealed that undermethylated GATC sequences were located exclusively within the single-stranded loop region of the sequence involved in H-DNA formation. Control molecules which either contained the pur-pyr tracts (pPPK and pKK42) or not (pUC18) and were not able to form the triple-stranded conformation were found to be normally methylated by the dam gene product in vivo. Location of GATC within the triplex forming sequence seems to be a prerequisite for achieving its in vivo undermethylation. E.coli host factors are involved in the observed phenomenon. This has been deduced from the fact that the undermethylated state of pPP1 and pPP2 does not depend on the phase of growth of host cells and is steadily maintained up to 50 hours, whereas the kinetics of Dam methylation in vitro of sites located within the triplex loop does not differ substantially from the kinetics of methylation of other sites on the vector. Full methylation can be readily achieved in vitro. Additional factor(s) that operate in vivo to control the undermethylated state are most likely proteins since the observed effect can be suppressed by chloramphenicol administration to the cell cultures.