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。尽管DNA聚合酶具有校正机制以最小化复制过程中的错误，但偶尔由于复制错误而导致的错配仍然发生。在所有的生物体中都有错配修复系统来防止这种突变的发生。E.大肠杆菌具有包括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。在过去的一年中，我们已经确定了MutH的晶体结构，MutH是一种229碱基序列特异性核酸内切酶，它在识别错配时被MutS激活。我们得到了MutH的两种晶型，并对两种晶型的MutH结构进行了解析和精细化。MutH的晶体结构使我们能够识别该酶的活性位点。然后我们进行了点突变以确认催化残基。基于晶体结构，我们推测MutH的激活机制。我们还鉴定了MutH与限制性内切酶如PvuII、EcoRV和Sau 3AI的结构相似性，并提出II型限制性内切酶是从一个共同的祖先进化而来的。我们现在已经确定了MutL的40 KdN-末端片段的晶体结构。MutL及其同源物，虽然是从细菌到人的DNA错配修复中不可或缺的，但在我们的结构表征之前还没有已知的功能。根据MutL与DNA促旋酶的一个含ATP酶的片段的结构同源性和我们从各种功能研究中获得的结果，我们认为MutL是一种ATP酶。我们已经证明ATP结合诱导MutL的构象变化，这种变化对于MutL在DNA修复中的功能是必不可少的。我们已经制造了各种缺乏ATP酶活性的突变体，目前正在进一步鉴定这些MutL突变体。为了继续DNA重组的研究，我们将注意力集中在细菌转座酶TN 10上。我们已经产生了大量的纯转座酶，IHF（DNA转座的辅因子）和各种DNA底物的晶体学研究的系统。脊椎动物V（D）J基因重排是免疫系统成熟的关键。它允许产生抗体和T细胞受体来建立防御系统。这种基因重排必须在细胞发育过程中受到严格控制。错误的重排常常导致基因截短或染色体易位，成为各种类型淋巴瘤的原因。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 Gellman博士团队首次证明了纯化的RAG-1和RAG-2蛋白可以在体外启动基因重排。小鼠主动RAG蛋白在昆虫细胞中得到过表达.本课题组检测了RAG蛋白在E.杆菌在制备了几十种不同的融合构建体后，我们终于成功地在E.这为这种极其重要的蛋白质的突变研究和晶体学研究铺平了道路。我们还克隆了人RAG蛋白，并构建了在大肠杆菌和昆虫细胞中表达的构建体。最终我们将利用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。- 晶体，分子结构，重组，DNA修复

Genes have to be replicated before every cycle of celldivision. Although DNA polymerase has a proofreading mechanism to minimize the errorsduring replication, occasionally mismatch due to replication- errors still happens. Inall living organisms there are mismatch repair systems to prevent such mutations fromoccurring. 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. During thelast year, we have determined crystal structures of MutH, a 229aasequence-specific endonuclease which is activated by MutS upon its recognition ofmismatch. We have obtained two crystal forms of MutH, and have solved and refinedMutH structures of bothcrystal forms. The crystal structure of MutH allow us toidentify the active site of thisenzyme. We then made point mutations to confirm the catalyticresidues. Based on the crystal structures, we postulate a mechanism for how MutH isactivated. We also identified the structual similarity between MutH and restrictionendonucleases, such asPvuII, EcoRV and Sau3AI and proposed that type II restrictionenzymes are evolved from a common ancestor. We have now determined the crystal structure of a 40KdN-terminal fragment of MutL. MutL and its homologues, although indispensable for DNAmismatch repair from bacterial to human, has no known function prior to our structuralcharacterization. Based on the structural homology of MutL to an ATPase-containing fragment ofDNA gyrase and results we obtained from various functional studies, we conclude thatMutL is an ATPase. We have since shown that ATP-binding induces conformational changes inMutL and such changes areessential for MutLs function in DNA repair. We have made variousmutants which lacksATPase activity and are currently further characerizing theseMutL mutants.To continue the studies of DNA recombination, we have focusedour attention ona bacterial transposase, TN10. We have generated ample amountsof pure transposase, IHF(a cofactor for DNA transposition) and various DNA substrates forcrystallographic studiesof the system. V(D)J gene rearrangement in vertebrates is essential for thematuration of immune systems. It allows the generation of antibodies and T-cellreceptors to build up the defense system. Such gene rearrangement has to be tightly controlledduring cell development. Erroneous rearrangement often leads to gene truncation orchromosome translocation that becomes causes of various types of lymphomas. V(D)J generearrangement 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 Gellerts group at NIH is the first todemonstrate purified RAG-1 and RAG-2 proteins can initiate gene rearrangement in vitro. ActiveRAG proteins from mouse have been over- expressed in insect cells. My group has testedexpression of RAG proteins in E. coli. After making dozens of different fusion constructs,we have finally succeeded in making active RAG-1 in E. coli, which paves the road for bothmutational studies as well as crystallographic studies of this extremely importantprotein. We have also cloned human RAG proteins and made constructs for expression in both E.coli and insect cells. Eventually we are going to determine the three-dimensionalstructures of RAG proteins and their complexes with the DNA recognition sequences using x-raycrystallographic techniques. - crystal, molecular structure, recombination, DNA repair