Elucidating Molecular Mechanisms of Cancer Development by Investigating Key DNA Repair Pathways

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Project Summary ! Dr. Sharonda LeBlanc earned her PhD in Nanoscale Science, investigating the effects of electric fields on single quantum dot (SQD) fluorescence emission using confocal microscopy coupled with time-resolved spectroscopy. She discovered interesting photophysical effects of moderate applied fields and plasmonic films on SQDs. Dr. LeBlanc is currently transitioning from fundamental physical chemistry to biochemistry/biophysics while still utilizing single molecule techniques. Currently, she is a postdoctoral researcher at UNC Chapel Hill investigating molecular interactions of mismatch repair proteins. She works between two labs at UNC (Chemistry) and NC State (Physics) to complete her research. Her career goal is to obtain a tenure-track position at a research institution. She would like to combine her past research experience in nanoscience with biochemistry/biophysics to ultimately investigate molecular mechanisms of cancer development in vivo. The specific aims of this proposal and research training plan are designed to enhance Dr. LeBlanc's skills and knowledge in the biological sciences, specifically DNA repair. Specific Aims 1 and 2 are as follows: Specific Aim 1: Characterize the nucleotide-dependent dynamics of MutL conformations in the absence of mismatch DNA in vitro using single molecule FRET. Specific Aim 2: Investigate the dynamics of wild-type and mutant MutL conformations in the context of mismatch repair initiation with nucleotides, MutS, and mismatch DNA in vitro with smFRET. DNA mismatch repair (MMR) is a post-replicative system of proteins that corrects rare mistakes in the genome of all organisms. In the human genome of 6 billion bases, there are ~ 600 errors per round of replication, per cell. If left uncorrected, errors accumulate as permanent mutations in a genome, and can lead to a disease state in the organism. MutS and MutL homologs are tasked with recognizing a mismatch in 107 correctly paired bases, discriminating between parent and daughter strand, then initiating repair. Single amino acid mutations in MutS and MutL proteins have been linked to hereditary and sporadic colorectal cancer, the third most common cancer worldwide. Although these mutations, mostly associated with MutL, have been identified in cancer cases, it is unclear how MMR deficiencies initiate and advance the disease. Failures in the mismatch repair pathway likely initiate tumorigenesis, but we lack a fundamental understanding of the MMR process. Single molecule fluorescence resonance energy transfer (smFRET) is uniquely capable of investigating the molecular mechanism of MMR that involves multiple transient protein-protein and protein-DNA interactions. These experiments may provide a basis for identifying therapeutic targets. The final aim of this proposal is designed to facilitate Dr. LeBlanc's transition to an independent career, outlined in the Research Strategy: Specific Aim 3: Develop ideas, design experiments, and test new hypotheses related to outstanding questions in DNA repair in preparation for an independent research career.