Analysis of FokI Cleavage Resistance Observed in DNA Sequences Generated via the Combinatorial Selection Method, REPSA
Date of Award
Master of Science in Chemical Sciences (MSCB)
Dr. Michael Van Dyke
Dr. Melanie Griffin
Dr. Daniela Tapu
FokI is a thoroughly investigated and highly utilized restriction endonuclease that recognizes the DNA sequence, 5’-GGATG-3’, and cleaves outside of this site 9 and 13 base-pairs downstream. The shifted cleavage function possessed by this kind of endonuclease is utilized in many applications including the combinatorial selection method, REPSA. FokI employment in the REPSA procedure has demonstrated the tendency to select for an unmodified sequence that possesses the recognition site yet is refractory to cleavage by the enzyme. Sequencing of the cleavage resistant DNA has revealed the inhibitory event to be induced by the presence of an additional inversely oriented recognition site. The work conducted here primarily aims to demonstrate FokI cleavage resistance in sequences bearing the identified motif and elucidate potential causative mechanisms. During this research, additional previously uncharacterized phenomena pertaining towards FokI dynamics were revealed. Through experimental observations aided by in-depth visual analysis of molecular models, potential mechanisms underlying observed activities were formulated that offer novel insight into the nature of FokI and the possible implications consequently emerging from this newfound understanding.