THE INFLUENCE OF PRIMARY AND SECONDARY DNA-STRUCTURE IN DELETION AND DUPLICATION BETWEEN DIRECT REPEATS IN ESCHERICHIA-COLI

We describe a system to measure the frequency of both deletions and du plications between direct repeats. Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within t he chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7. This creates an insert between direct repeated EcoRI sites and result s in a chloramphenicol-sensitive phenotype. Selection for chlorampheni col resistance was utilized to select chloramphenicol resistant revert ants that included those with precise deletion of the insert from plas mid pBR325 and duplication of the insert in plasmid pJT7. The frequenc y of deletion or duplication varied more than 500-fold depending on th e sequence of the short sequences inserted into the EcoRI site. For th e nonpalindromic inserts, multiple internal direct repeats and the len gth of the direct repeats appear to influence frequency of deletion. C ertain palindromic DNA sequences with the potential to form DNA hairpi n structures that might stabilize the misalignment of direct repeats h ad a high frequency of deletion. Other DNA sequences with the potentia l to form structures that might destabilize misalignment of direct rep eats had a very low frequency of deletion. Duplication mutations occur red at the highest frequency when the DNA between the direct repeats c ontained no direct or inverted repeats. The presence of inverted repea ts dramatically reduced the frequency of duplications. The results sup port the slippage-misalignment model, suggesting that misalignment occ urring during DNA replication leads to deletion and duplication mutati ons. The results also support the idea that the formation of DNA secon dary structures during DNA replication can facilitate and direct speci fic mutagenic events.