NOVEL POST-REPLICATIVE DNA MODIFICATION IN STREPTOMYCES - ANALYSIS OFTHE PREFERRED MODIFICATION SITE OF PLASMID PIJ101

Both Streptomyces lividans and Streptomyces avermitilis have the abili ty to site specifically modify their DNA, rendering it susceptible to in vitro Tris-dependent double-strand cleavage, We have cloned a 160 b p fragment containing the preferred modification site of plasmid pIJ10 1 and, employing an in vitro primer extension assay, determined that t he modifications occur at guanine residues on either strand separated by 3 bp, These guanines are located within a 6 bp palindromic 'core' s equence, A cloned copy of a 35 bp region of the plasmid containing thi s core sequence was not recognized by the modifying activity in vivo, To further investigate the nature of the site specificity a set of del etion mutants of the 160 bp sequence were analysed, This revealed that a substantial portion of this sequence is essential for authentic mod ification. The essential region contains three 13 bp direct repeats, t he central one containing the core sequence, while the left-hand and r ight-hand copies overlap two potential stem-loop structures, Deletion of either left- or right-hand repeat structures abolishes modification within the core sequence, although the left-hand deletion resulted in modification at a secondary site within the right-hand direct repeat. These data support a postreplicative mechanism of modification, under lined by the observation that the modifications are not detected in si ngle-stranded plasmid replication intermediates.