Mismatch Repair Proteins to Antibody Class Switching

描述（由申请方提供）：长期目的：表征抗体类别转换重组中错配修复蛋白的机制。这对于理解优化抗体功能的机制是必要的，以充分和安全地对抗病原体感染，同时保持基因组完整性和预防恶性肿瘤。已知错配修复基因产物参与类别转换。从错配修复机制的既定特征，可以提出一个模型，其中错配修复蛋白识别开关区DNA中间体，并刺激DNA切割，这是必要的类转换通过各种途径。该模型可以通过错配修复蛋白结合并刺激代表重组中间体的开关区DNA的切割的假设来实验性地解决。具体目的1：证明重组错配修复蛋白直接结合免疫球蛋白开关重组DNA中间体。将通过电迁移率变动测定来测量六种开关区底物与三种蛋白质复合物之间的相对亲和力。具体目的2：显示错配修复蛋白刺激免疫球蛋白开关重组DNA中间体的切割。然后在体外切割测定中测试这些蛋白质，以测试它们刺激切割的能力和激活的B细胞核提取物中核酸酶对开关区底物的加工。活化B细胞核提取物中的错配修复蛋白诱导的溶核活性将是蛋白质分级分离并通过质谱表征。

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.