Double-strand breaks in plasmid DNA and the induction of deletions

Abstract

Double-strand breaks (dsbs) have been produced in plasmid DNA by various restriction endonucleases and the survival and the deletion mutation incidence have been measured in E. coli. The deletion formation is known to depend upon the occurrence of short direct repeats within the DNA molecule. In order to study the role of these repeats we constructed plasmid molecules with repeats of various lengths or with a 10-base pair repeat at different distances from each other. Furthermore the influence of the location and the structure of the dsb was studied. Repair and deletion frequencies of the linearized plasmids were measured after transformation of E. coli. The yield of the specific deletion mutation (the one which occurs between the introduced repeats) increases nearly linearly with the square of the length of the repeat, while the yield of the correctly repaired DNA and the yield of all other deletion mutants remained constant. The slope of the linear increase of the yield of the specific deletion depends on the location and the structure of the dsb. The yield of the specific deletion mutation decreases with increasing distance between the repeats. A proposal for the rate-determining step of the deletion formation is made.