Investigating the mechanisms of action of glycosylases and nucleases using classical and quantum mechanical computational techniques

Abstract

The phosphodiester and glycosidic bonds are essential for the function of nucleic acids, binding nucleotides together and nucleobases to (deoxy)ribose. However, many cellular processes require cleaving these bonds, including DNA repair, RNA processing, and viral defense. To enable these processes to occur at biologically-relevant timescales, cells utilize glycosylases and nucleases to facilitate this chemistry. Due to their important roles in cell survival, glycosylases and nucleases are implicated in many different diseases and disorders, including neurodegeneration, inflammation, and cancer. Despite this, the catalytic mechanism for many of these enzymes is poorly understood, with multiple proposals often present in the literature. In this thesis, computational methodologies, including molecular dynamics (MD) simulations, free energy techniques, quantum mechanics/molecular mechanics (QM/MM) calculations, and QM/MM MD simulations were used to clarify the atomic level details of glycosylase and nuclease catalytic mechanisms. Specifically, the monofunctional glycosylases MutY, MBD4, and AlkA were investigated, focusing on their potential to invoke a novel mechanism that involves a DNA−protein crosslinked intermediate. To complement the work on monofunctional glycosylases, the bifunctional glycosylase hOGG1 was also studied, with this work representing the first characterization of a bifunctional glycosylase β-lyase mechanism. Finally, phosphodiester bond cleavage was further investigated by studying the Dicer mechanism of action, with the elucidated mechanism used to gain insight into the disease-causing nature of DICER1 hotspot mutants. The improved understanding of nuclease and glycosylase mechanisms will allow for the rational design of small molecule inhibitors as therapeutics and pushes forward the use of these enzymes for biotechnological applications.