STRUCTURAL STUDIES OF DNA RECOMBINATION AND MISMATCH REPAIR

基因必须在每个细胞分裂周期之前复制。虽然DNA聚合酶具有校正机制以最小化复制过程中的错误，但偶尔仍会发生由于复制错误而导致的错配。在所有生物体中，都有错配修复系统来防止这种突变的发生。 E.大肠杆菌具有包含MutS、MutL和MutH蛋白的甲基指导的错配修复系统。MutS和MutL蛋白的同源物也在人类中发现。这些蛋白质的突变在90%的遗传性非息肉病性大肠癌中被鉴定。在1999年10月之前的两年中，我们的研究小组确定了错配修复蛋白MutH和MutL的一个保守的40 KD片段的晶体结构，发现了MutL的一个固有的ATP酶活性，这是科学家们多年来一直没有发现的，并通过诱变和生物化学手段进行了功能研究。我们最近测定了190 KdTaqMutS单独、与DNA复合以及与DNA和ADPMg 2+的三元复合物的晶体结构。 这些晶体结构揭示了：（1）MutS蛋白的结构，（2）MutS如何结合未配对碱基，（3）MutS如何识别广泛的“错配”而没有序列特异性，（4）ATP结合位点的组分和水解机制，（5）ATP酶和错配结合活性如何在MutS中协调，以及（6）人类MSH蛋白中的突变导致癌症的“热点”。为了继续研究DNA重组，我们继续了V（D）J重组的研究。 脊椎动物V（D）J基因重排对免疫系统的成熟至关重要。 它允许产生抗体和T细胞受体来建立防御系统。 这种基因重排在细胞发育过程中必须受到严格控制。错误的重排往往导致基因截短或染色体易位，成为各种类型的淋巴瘤的原因。 V（D）J基因重排是一种位点特异性DNA重组。 RAG-1和RAG-2（重组激活基因产物）是启动基因重排的必要条件。 美国国立卫生研究院的Martin Gellert博士的研究小组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研究小组。 小鼠RAG活性蛋白在昆虫细胞中得到过表达。我的小组已经测试了RAG蛋白在大肠杆菌中的表达。杆菌在E.大肠杆菌，与马萨诸塞州的Marjorie Oettinger博士合作。综合医院，我们已经发现了三个保守的酸性残基负责RAG 1蛋白的DNA切割活性，并构成活性位点。 我们还克隆、表达和纯化了RAG 1蛋白的各种片段用于晶体学目的。最终，我们将使用X射线晶体学技术确定RAG蛋白及其与DNA识别序列的复合物的三维结构。

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.