Abstract
Spontaneous mutations naturally occur for a variety of reasons throughout the Escherichia coli genome. These mutations are the fundamental method of diversity and change throughout all organisms. Of particular importance are the mutations that arise during template-switching at the site of short repetitive sequences in DNA. These sequences are known as quasi-palindromes. Using a variety of approaches, this study sought to better understand where mutational hotspots and quasi-palindromes are located in the E. coli genome. A series of knockout strains were used in which the mismatch repair (MMR) system was disrupted. Without mutS, it was found that mutations accumulate at a much higher frequency throughout the entire E. coli genome. Without mutS, xonA, and xseA, fewer mutations accumulated, but a quasi-palindrome within the rpoN gene was located and sequence confirmed. The disruption of the MMR system led to mutations dominated by transitions at much higher rates than in wild-type. This study indicated the significance of mutS for its role in mismatch repair and template-switch mutations. A novel protocol was created that allowed the precise excision of quasi-palindrome secondary structures from genomic E. coli DNA. This protocol utilized the ability of the quasi-palindromes to form secondary structures as a unique way to isolate them from normal dsDNA and ssDNA in vitro. This protocol will give scientists the tools to locate mutational hotspots within genomic DNA and better understand mechanisms of template-switch mutations.