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不匹配。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。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。在过去的一年里，我们已经确定了Muth的晶体结构，Muth是一种序列特异性内切酶，在识别错配后被MutS激活。我们得到了MuTh的两种晶型，并对这两种晶型的MutH结构进行了求解和改进。Muth的晶体结构使我们能够确定该酶的活性部位。然后，我们进行了点突变以确认催化残基。基于晶体结构，我们假设了Muth如何被激活的机制。我们还鉴定了Muth与限制性内切酶PvuII、EcoRv和Sau3AI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40KdN末端片段的晶体结构。MutL及其同源物虽然对于细菌到人类的DNA错配修复是必不可少的，但在我们的结构表征之前没有已知的功能。根据MutL与DNA旋转酶的ATPase片段的结构同源性，以及我们在各种功能研究中获得的结果，我们得出结论：MutL是一种ATPase。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。我们已经培育了各种缺乏ATPase活性的突变体，目前正在进一步鉴定这些突变体。为了继续DNA重组的研究，我们将注意力集中在细菌转座酶Tn10上。我们已经产生了大量的纯转座酶、IHF(DNA转座的辅助因子)和各种DNA底物，用于系统的结晶学研究。脊椎动物的V(D)J基因重排对免疫系统的成熟起着至关重要的作用。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遗传排列是一种位点特异性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。美国国立卫生研究院的马丁·盖勒茨博士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人。小鼠ActiveRAG蛋白已在昆虫细胞中过表达。我的团队测试了RAG蛋白在大肠杆菌中的表达。经过几十种不同的融合构建体，我们终于成功地在大肠杆菌中制备了活性的RAG-1，这为这一极其重要的蛋白质的突变研究以及结晶学研究铺平了道路。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结构。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识别序列的络合物的三维结构。-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