Abstract
Accurate DNA replication is essential for faithful transmission of hereditary information throughout all kingdoms of life. A mistake in DNA replication can lead to accumulation of mutations that are often deleterious to the life of an organism. Eliciting mechanisms in which mutations arise is vital for disease prevention and treatment. Mutational hotspots exist at imperfect inverted repeats, quasi-palindromes, which get perfected by mutagenesis. Given the “patchy” nature of quasi-palindromic sequences it is quite difficult to identify them bioinformatically and therefore quasi-palindrome associated mutations have remained an understudied class. The proximity of inverted repeats makes it possible for newly synthesized DNA to misalign and form a complementary hairpin that polymerases can incorrectly use as a template. Template-switch mutagenesis has been implicated in cancer and Duchenne muscular dystrophy. Replication proteins are highly conserved from bacteria to humans allowing for the Escherichia coli model system to be used in illumination of quasi-palindrome mutational avoidance and its stimulation by pharmacological mutagens. The work described quantifies the activity of Exonuclease I and Exonuclease VII in quasi-palindrome premutagenic avoidance using a novel template-switch specific E. coli strain reporter in lacZ. The activity of the Exonuclease I is in a strand-specific manner, assisting avoidance of mutation on the lagging strand. This work also uncovered that 5-azacytidine, an FDA-approved cancer treatment drug, stimulates quasi-palindrome events. The aim is to uncover how quasi-palindrome mutations are avoided and stimulated, potentially leading to improvements in clinical treatments or prevention.