STRUCTURAL STUDIES OF DNA RECOMBINATION AND MISMATCH REPAIR

基因必须在细胞分裂的每个周期之前复制。虽然DNA聚合酶有一种校对机制来最大限度地减少复制过程中的错误，但偶尔仍会发生由于复制错误而导致的错配。在所有活着的生物体中，都有错配修复系统来防止这种突变的发生。大肠杆菌有一个甲基导向的错配修复系统，包括MutS、MutL和Muth蛋白。在人类中也发现了MutS和MutL蛋白的同源物。这些蛋白的突变在90%的遗传性非息肉性结直肠癌中被发现。在1999年10月之前的两年里，我们的团队确定了错配修复蛋白MuTH的晶体结构和MutL的一个保守的40KD片段，发现了MutL的内在ATPase活性，这是科学家们多年来一直不知道的，并通过突变和生化手段进行了功能研究。最近，我们与NIDDK的谢佩吉博士的团队合作，确定了190kD的Taq MutS的晶体结构，它单独与DNA形成络合物，并作为DNA和ADPMg2+的三元络合物。这些晶体结构揭示了(1)MutS蛋白的结构，(2)MutS如何结合未配对的碱基，(3)MutS如何在没有序列特异性的情况下识别广泛的“错配”，(4)ATP结合位点的组成和水解机制，(5)ATPase和错配结合活性在MutS中是如何协调的，以及(6)人类MSH蛋白中突变导致癌症的“热点”。为了继续DNA重组的研究，我们继续了对V(D)J重组的研究。脊椎动物的V(D)J基因重排是免疫系统成熟所必需的。它允许产生抗体和T细胞受体来建立防御系统。在细胞发育过程中，这种基因重排必须受到严格控制。错误的重排往往会导致基因截断或染色体易位，从而导致各种类型的淋巴瘤。V(D)J基因重排是一种定点DNA重组。两种蛋白质，RAG-1和RAG-2(重组激活基因产物)是在体内启动基因重排的必要条件和充分条件。NIH的Martin Gellert博士的团队是第一个证明纯化的RAG-1和RAG-2蛋白可以在体外启动基因重排的小组。小鼠RAG活性蛋白已在昆虫细胞中过表达。我的团队已经测试了RAG蛋白在大肠杆菌中的表达。在与马萨诸塞州的玛乔里·厄廷格博士合作，最终成功地在大肠杆菌中制造了活性RAG-1之后。我们发现这三个保守的酸性残基与RAG1蛋白的DNA切割活性有关，并构成活性部位。我们还克隆、表达和纯化了RAG1蛋白的各种片段，用于结晶学目的。最终，我们将使用X射线结晶学技术来确定RAG蛋白及其与DNA识别序列的复合体的三维结构。

Genes have to be replicated before every cycle of cell division. Although DNA polymerase has a proofreading mechanism to minimize the errors during replication, occasionally mismatch due to replication-errors still happens. In all living organisms there are mismatch repair systems to prevent such mutations from occurring. E. coli has a methyl-directed mismatch repair system comprising MutS, MutL and MutH proteins. Homologues of MutS and MutL proteins are also found in human. Mutations in these proteins are identified in 90% of the hereditary nonpolyposiscolorectal cancers. In the two years before Oct. 1999, our group determined the crystal structures of mismatch repair proteins MutH and a conserved 40KD fragment of MutL, discovered an intrinsic ATPase activity of MutL, which had eluded scientists for years, and pursued functional studies by mutagenesis and biochemical means.In collaboration with Dr. Peggy Hsieh's group at NIDDK, we have recently determined the crystal structures of the 190 Kd Taq MutS alone, complexed with DNA, and as a ternary complex with DNA and ADPMg2+. These crystal structures have revealed, (1) the architecture of MutS proteins, (2) how MutS binds an unpaired base, (3) how MutS recognizes a broad range of 'mismatches' without sequence specificity, (4) the components of the ATP binding site and hydrolysis mechanism, (5) how the ATPase and the mismatch binding activities are coordinated in MutS, and (6) the 'hot spots' in human MSH proteins where mutations lead to cancers. To continue the studies of DNA recombination, we have continuted our research on V(D)J recombination. V(D)J gene rearrangement in vertebrates is essential for the maturation of immune systems. It allows the generation of antibodies and T-cell receptors to build up the defense system. Such gene rearrangement has to be tightly controlled during cell development. Erroneous rearrangement often leads to gene truncation or chromosome translocation that becomes causes of various types of lymphomas. V(D)J gene rearrangement is a type of site-specific DNA recombination. Two proteins, RAG-1 and RAG-2 (recombination activation gene products), are necessary and sufficient to turnon the gene rearrangement in vivo. Dr. Martin Gellert's group at NIH is the first to demonstrate purified RAG-1 and RAG-2 proteins can initiate gene rearrangement in vitro. Active RAG proteins from mouse have been over-expressed in insect cells. My group has tested expression of RAG proteins in E. coli. After finally succeeding in making active RAG-1 in E. coli, in collaboration with Dr. Marjorie Oettinger at Mass. General Hospital, we have found the three conserved acidic residues to be responsible for DNA cleavage activity of RAG1 protein and constitute the active site. We have also cloned, expressed and purified various fragments of RAG1 protein for crystallographic purpose. Eventually we are going to determine the three-dimensional structures of RAG proteins and their complexes with the DNA recognition sequences using x-ray crystallographic techniques.