Mismatch Repair Proteins to Antibody Class Switching

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DESCRIPTION (provided by applicant): Long-term objective: To characterize the mechanisms of mismatch repair proteins in antibody class switch recombination. This is necessary for understanding the mechanisms of optimizing antibody function to adequately and safely counter pathogen infection while maintaining genomic integrity and preventing malignancy. Mismatch repair gene products are known to be involved in class switching. From established characteristics of mismatch repair mechanisms, a model can be proposed in which mismatch repair proteins recognize switch region DNA intermediates and stimulate DNA cleavage that is necessary for class switching by various pathways. This model can be experimentally addressed by the hypothesis that mismatch repair proteins bind to and stimulate cleavage of switch region DNA representative of recombination intermediates. Specific Aim 1: To demonstrate that recombinant mismatch repair proteins directly bind immunoglobulin switch recombination DNA intermediates. The relative affinity between six switch region substrates and three protein complexes will be measured by electromobility shift assay. Specific Aim 2: To show that mismatch repair proteins stimulate cleavage of immunoglobulin switch recombination DNA intermediates. These proteins will then be tested in an in vitro cleavage assay to test their ability to stimulate cleavage and processing of the switch region substrates by nucleases in activated B cell nuclear extracts. Mismatch repair protein-induced nucleolytic activities in activated B cell nuclear extracts will be protein fractionation and characterized by mass spectrometry.