| Abstract: | The frequencies of deletion of short sequences (mutation inserts) inserted into the chloramphenicol acetyl-transferase (CAT) gene were measured for pBR325 and pBR523, in which the orientation of the CAT gene was reversed, in Escherichia coli. Reversal of the CAT gene changes the relationship between the transcribed strand and the leading and lagging strands of the DNA replication fork in pBR325-based plasmids. Deletion of these mutation inserts may be mediated by slipped misalignment during DNA replication. Symmetrical sequences, in which the same potential DNA structural misalignment can form in both the leading and lagging strands, exhibited an approximately twofold difference in the deletion frequencies upon reversal of the CAT gene. Sequences that contained an inverted repeat that was asymmetric with respect to flanking direct repeats were designed. With asymmetric mutation inserts, different misaligned structural intermediates could form in the leading and lagging strands, depending on the orientation of the insert and/or of the CAT 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 leading strand. These results support the model that slipped misalignment involving DNA secondary structure occurs preferentially in the lagging strand during DNA replication. |
