Structural biology of human DNA mismatch repair machinery

DNA错配修复(MMR)是基因组稳定的主要因素。MMR纠正DNA 生物合成错误，确保基因重组的保真度，是细胞 对某些类别的DNA损伤的反应，包括由几种 化疗药物(如顺铂)。MMR在体细胞过度突变中也起着重要作用 免疫球蛋白多样性的产生。人类MMR的缺陷与一种 肿瘤易感性强，CTG-CAG重复序列突变扩增 几种神经肌肉疾病(亨廷顿氏病)的致病突变序列 疾病、脆性X综合征和强直性肌营养不良)。尽管这一系统在 人类的健康和疾病，我们对其分子机制的了解是有限的。我们建议 将X射线结晶学、溶液小角X射线散射(SAXS)和生化相结合 研究人类蛋白质-DNA组件的方法，这些组件是病变中的关键中间体 MMR的识别和切除步骤。我们很幸运，人类MMR途径现在已经 达到了分析的成熟度，获得蛋白质组分及其晶体现在是 手，可以直接研究。我们已经确定了人类MutS的晶体结构 DNA损伤识别复合体。在目标1中，我们建议确定Exo1的晶体结构 和MutS？在目标2中，我们研究了底物识别、特异性和ATP依赖 MMR组分的构象转变。在目标3中，我们研究了两个关键的多组件 对识别和切除过程至关重要的程序集。这些目标的共同作用将有助于 进一步加深我们对MMR相关人类疾病的分子基础的理解。

DNA mismatch repair (MMR) is a major contributor to genome stability. MMR corrects DNA biosynthetic errors, ensures the fidelity of genetic recombination and is required for the cellular response to certain classes of DNA damage, including lesions induced by several chemotherapeutic drugs (e.g. cisplatin). MMR also has an essential role in somatic hypermutation for the generation of immunoglobin diversity. Defects in human MMR are associated with a strong predisposition to tumor development, and mutagenic expansion of CTG-CAG repeat sequence which are the causative mutations for several meuromuscular diseases (Huntington's disease, fragile-X syndrome, and myotonic dystrophy). Despite the importance of this system in human health and disease, our understanding of its molecular mechanisms is limited. We propose to combine X-ray crystallography, solution small angle X-ray scattering (SAXS) and biochemical approaches to study the human protein-DNA assemblies that are key intermediates in the lesion recognition and excision steps of MMR. We are fortunate that the human MMR pathway has now reached the maturity of analysis that obtaining the protein components and their crystals is now in hand and can be studied directly. We have already determined crystal structures of human MutS¿ DNA lesion recognition complexes. In Aim 1 we propose to determine crystal structures of Exo1 and MutS¿. In Aim 2 we investigate substrate recognition, specificity and ATP-dependent conformational transitions of MMR components. In Aim 3 we study two key multi-component assemblies essential to the recognition and excision process. Together the aims will contribute to furthering our understanding of the molecular basis of MMR-associated human diseases.