Mismatch Repair Proteins to Antibody Class Switching

描述(由申请人提供)：长期目标：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的机制是必要的。错配修复基因产物已知与类别转换有关。根据错配修复机制的已有特征，可以提出一个模型，在该模型中，错配修复蛋白识别开关区DNA中间体，并通过各种途径刺激DNA切割，这是进行类别转换所必需的。这一模型可以通过错配修复蛋白与代表重组中间产物的开关区DNA结合并刺激其切割的假设来实验解决。具体目的1：证明重组错配修复蛋白直接与免疫球蛋白开关重组DNA中间体结合。通过电迁移率位移分析，可以测量6种开关区域底物与3种蛋白质复合体的相对亲和力。特定目的2：证明错配修复蛋白刺激免疫球蛋白开关重组DNA中间体的裂解。然后，这些蛋白质将在体外切割试验中进行测试，以测试它们通过激活的B细胞核提取液中的核酸酶刺激开关区域底物的切割和加工的能力。错配修复蛋白在活化的B细胞核提取液中诱导的核溶解活性将被蛋白质分级，并用质谱仪进行表征。

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.