Overlapping direct repeats stimulate deletions in specially designed derivatives of plasmid pBR325 in Escherichia...

Current misalignment mutagenesis models have identified certain sequences such as direct and inverted repeats, which can stabilize transient misalignments on single-stranded DNA, as major structural parameters for deletions. We have constructed derivatives of the plasmid pBR325 to investigate further the relative roles of such sequences under controlled conditions. The plasmid derivatives pOCE15, pRS1 and pRS4, were obtained by cloning fragments of the same approximate size (60-64 bp) but different sequence into the unique EcoR1 site of the chloramphenicol acetyl transferase (cat) gene of plasmid pBR325. The insert of pOCE15 is a perfectly palindromic lac operator fragment. Both pRS1 and pRS4 carry the same non-palindromic fragment but differ from each other in the sequence at the right (3') end of the insert. Plasmid pRS4 differs from pRS1 by a 9-bp duplication containing an additional EcoR1 site at the 3' end of the insert. This arrangement yields overlapping imperfect 17-18 bp and perfect 11 bp direct repeats at the deletion termini and creates multiple opportunities for the stabilization of misaligned intermediates. The deletion rate, measured from the reversion of chloramphenicol sensitivity (Cms) to resistance (Cmr), was always highest in pRS4, intermediate in pOCE15 and lowest in pRS1, with an approximate ratio of 100:10:1. We have also obtained evidence for the participation of RecA in the genesis of deletions in these pBR325-derived plasmids.