Molecular mechanisms of modular nuclease domains

Project Summary/Abstract Nucleases are a class of enzyme that hydrolyze nucleic acid substrate in a variety of cellular processes. Their activity is a requirement for the maintenance of genomic integrity in DNA replication, repair, and recombination. Understanding nuclease regulation and specificity in these processes is critical for modeling these fundamental pathways and human diseases linked to dysregulation. This includes Lynch syndrome, a cancer predisposition syndrome linked to colorectal and endometrial cancers, Huntington’s disease, and infertility. Nucleases unique to bacteria are also potential targets for antibiotics and a complete understanding of nuclease biochemistry paves the way for the discovery of new drug targets and exploiting nucleases as new biotechnological tools and therapeutic agents. Characterizing and developing novel nucleases is a future area of interest for my research program. We will use proteins in DNA repair processes to model nuclease activity and determine regulation and specificity steps. Using the tractable DNA mismatch repair pathway which spellchecks newly replicated DNA, we will identify how inherently nonspecific nucleases can be given specificity. Proteins in this process have been co-opted for meiotic recombination and also play a role in the regulation of trinucleotide repeat expansions indicating that the associated nuclease activity is modular. This work addresses the origins of this co-option and provides missing mechanistic detail for how all of these pathways communicate substrate specificity to nucleolytic sites. In bacteria, homologous recombination is a method for acquiring antibiotic resistance. MutS2, a homolog to mismatch recognition complexes, has been implicated in several bacteria as being involved in this pathway. Its mechanisms of action and whether it follows paradigms established by canonical mismatch repair proteins are not clear and are addressed by mechanistic work described here. The nuclease domain of MutS2 is found throughout biology as a fusion to proteins with diverse specificities and functions. We will determine the modularity of this nuclease domain, how it achieves specificity by other domains, and test its potential for adaptation as a gene editing tool. We will also investigate the specificity and regulatory mechanisms of the newly discovered NucS protein which is multi-functional, and is implicated in multiple DNA repair processes in archaea and mycobacteria, including Mycobacterium tuberculosis, the bacteria that causes tuberculosis. This will provide key evidence for adaptation processes of organisms that utilize NucS in DNA repair processes. Our work will provide an underpinning for complex mechanistic models that can be ultimately used to detect and develop therapeutics for human disease. In addition, this work provides general insight into how nucleases are regulated and will guide future studies in other cellular pathways.