DOUBLE-STRAND BREAKS IN PLASMID DNA AND THE INDUCTION OF DELETIONS

Double-strand breaks (dsbs) have been produced in plasmid DNA by vario us restriction endonucleases and the survival and the deletion mutatio n incidence have been measured in E. coli. The deletion formation is k nown to depend upon the occurrence of short direct repeats within the DNA molecule. In order to study the role of these repeats we construct ed plasmid molecules with repeats of various lengths or with a 10-base pair repeat at different distances from each other. Furthermore the i nfluence of the location and the structure of the dsb was studied. Rep air and deletion frequencies of the linearized plasmids were measured after transformation of E. coli. The yield of the specific deletion mu tation (the one which occurs between the introduced repeats) increases nearly linearly with the square of the length of the repeat, while th e yield of the correctly repaired DNA and the yield of all other delet ion mutants remained constant. The slope of the linear increase of the yield of the specific deletion depends on the location and the struct ure 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.