DIFFERENTIAL DNA SECONDARY STRUCTURE-MEDIATED DELETION MUTATION IN THE LEADING AND LAGGING STRANDS

The frequencies of deletion of short sequences (mutation inserts) inse rted into the chloramphenicol acetyltransferase (CAT) gene were measur ed for pBR325 and pBR523, in which the orientation of the CAT gene was reversed, in Escherichia coli. Reversal of the CAT gene changes the r elationship between the transcribed strand and the leading and lagging strands of the DNA replication fork in pBR325-based plasmids. Deletio n of these mutation inserts may be mediated by slipped misalignment du ring DNA replication. Symmetrical sequences, in which the same potenti al DNA structural misalignment can form in both the leading and laggin g strands, exhibited an approximately twofold difference in the deleti on frequencies upon reversal of the CAT gene. Sequences that contained an inverted repeat that was asymmetric with respect to flanking direc t repeats were designed. With asymmetric mutation inserts, different m isaligned structural intermediates could form in the leading and laggi ng strands, depending on the orientation of the insert and/or of the C AT gene. When slippage could be stabilized by a hairpin in the lagging strand, thereby forming a three-way junction, deletion occurred by up to 50-fold more frequently than when this structure formed in the lea ding strand. These results support the model that slipped misalignment involving DNA secondary structure occurs preferentially in the laggin g strand during DNA replication.