Structural and functional diversity of the methyl-binding domain protein family

描述(由申请人提供)：该项目的长期目标是确定不同的MBD蛋白靶向特定的甲基化DNA序列以执行不同功能角色的分子机制。在哺乳动物中，MBD蛋白家族至少由7种蛋白质组成，涉及正常和病理发育基因沉默、DNA错配修复和癌变过程中肿瘤抑制基因的异常沉默。在研究Mbd2与甲基化DNA结合的分子细节时，我们已经证明该蛋白可以在同一DNA分子中的结合位置之间快速交换。这一观察结果使我们假设，不同的功能角色至少部分取决于所涉及的特定MBD的动态和结构DNA结合特征。在此观察的基础上，我们计划在多个靶点的背景下研究MBDS与DNA的序列依赖和独立结合。特别是，我们将重点介绍Mbd2、MBD3和MBD4的结构和结合亲和力。虽然Mbd2和MBD3高度同源，但Mbd2与甲基化DNA有更高的亲和力和选择性。相反，MBD4识别由甲基胞嘧啶的水解性脱氨基引起的G-T碱基对错配。拟议的工作将涉及核磁共振光谱、表面等离子激元共振、等温滴定量热法和荧光各向异性技术。我们将把这些生物物理和结构分析的结果与组织培养细胞比较的研究联系起来。 Mbd2突变体和结构域互换嵌合体的功能。后一项研究将确定不同MBD蛋白的功能特异性是否反映了MBD本身的DNA结合和结构特性。这些实验将涉及共IP、芯片和组织培养细胞中的基因表达研究。最终，这项工作将为DNA甲基化依赖的基因沉默和DNA错配修复的机制提供独特的见解。

DESCRIPTION (provided by applicant): The long-term objective of this project is to determine the molecular mechanisms by which different MBD proteins target specific methylated DNA sequences to carry out distinct functional roles. In mammals, the MBD protein family consists of at least seven proteins involved in different aspects of normal and pathologic developmental gene silencing, DNA mismatch repair, and aberrant tumor suppressor gene silencing in carcinogenesis. In studying the molecular details of MBD2 bound to methylated DNA, we have shown that the protein can exchange rapidly between binding sites in the same DNA molecular. This observation led us to postulate that the distinct functional roles depend, at least in part, o dynamic and structural DNA binding characteristics of the particular MBD involved. Building on this observation, we plan to investigate the sequence dependent and independent binding of MBDs to DNA in the context of multiple target sites. In particular, we will focus on the structure and binding affinity of MBD2, MBD3, and MBD4. Although MBD2 and MBD3 are highly homologous, MBD2 binds with greater affinity and selectivity for methylated DNA. In contrast, MBD4 recognizes a G-T base pair mismatch arising from hydrolytic deamination of methyl-cytosine. The proposed work will involve NMR spectroscopy, surface plasmon resonance, isothermal titration calorimetry, and fluorescence anisotropy techniques. We will correlate the results of these biophysical and structural analyses with studies in tissue culture cells comparing the function of MBD2 mutants and domain swap chimeras. The latter studies will determine whether functional specificity of different MBD proteins reflects the DNA binding and structural properties of the MBD itself. These experiments will involve co-IP, ChIP, and gene expression studies in tissue culture cells. Ultimately, this work will provide unique insight into the mechanisms of DNA methylation dependent gene silencing and DNA mismatch repair.