Roles of Double-strand Breaks, Nicks, and Gaps in Stimulating Deletions of CTG·CAG Repeats by Intramolecular DNA Repair

A series of plasmids harboring CTG·CAG repeats with double-strand breaks (DSB), single-strand nicks, or single-strand gaps (15 or 30 nucleotides) within the repeat regions were used to determine their capacity to induce genetic instabilities. These plasmids were introduced into Escherichia coli in the presence of a second plasmid containing a sequence that could support homologous recombination repair between the two plasmids. The transfer of a point mutation from the second to the first plasmid was used to monitor homologous recombination (gene conversion). Only DSBs increased the overall genetic instability. This instability took place by intramolecular repair, which was not dependent on RuvA. Double-strand break-induced instabilities were partially stabilized by a mutation in recF. Gaps of 30 nt formed a distinct 30 nt deletion product, whereas single strand nicks and gaps of 15 nt did not induce expansions or deletions. Formation of this deletion product required the CTG·CAG repeats to be present in the single-stranded region and was stimulated by E. coli DNA ligase, but was not dependent upon the RecFOR pathway. Models are presented to explain the intramolecular repair-induced instabilities and the formation of the 30 nt deletion product.