RUI: Refractory Cleavage Sites to Elucidate Type IIS Restriction Endonuclease Mechanisms

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With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Professor Michael Van Dyke from Kennesaw State University (KSU) to investigate an enzymes called type IIS restriction endonucleases that cleaves deoxyribonucleic acid (DNA) in specific places. Restriction endonucleases are critical tools in biotechnology, allowing investigators to make specific fragments of DNA. The fragments can be recombined to create new DNA sequences, otherwise known as recombinant DNA. Type IIS restriction endonucleases, recognize specific DNA sequences, but then cleave DNA at fixed distances away from them. Previous work reported that this cleavage occurred without any regard to the specific sequence (sequence specificity). However, sequences have now been identified that are completely resistant to this cleavage. This project investigates why certain DNA sequences are unable to be cleaved by type IIS restriction endonucleases, which may impact their use in biotechnology. Also, this research incorporates a mix of undergraduate and Master's graduate students, providing them with training in the biochemistry of nucleic acids and associated enzymes. Professor Van Dyke mentors graduate school-oriented undergraduate students and maintenance of the nascent Sequencing Core Facility at KSU.  This research project is undertaken to mechanistically determine why specific DNA sequences are refractory to the type IIS restriction endonuclease FokI. Cleavage-resistant sequences identified through the combinatorial approach Restriction Endonuclease Protection, Selection, and Amplification are sequenced and resistant motifs identified for Motif Elicitation analysis. These sequences are independently validated using an internally controlled Restriction Endonuclease Protection Assay to characterize observed cleavage resistance as a function of time, temperature, and concentration. Stopped-flow spectroscopy studies are then performed using Förster resonance energy transfer (FRET) to dissect individual steps during the process of DNA cleavage by FokI and to determine those affected by the identified cleavage-resistant sequences. Information from this study may provide a better mechanistic understanding of DNA recognition and cleavage by IIS restriction endonucleases, with potential applicability to other DNA cleavage/modification systems.