STRUCTURAL STUDIES OF DNA RECOMBINATION AND REPAIR

脊椎动物的V(D)J基因重排是其成熟所必需的 免疫系统。它允许产生抗体和T细胞 受体来建立防御系统。这种基因重排已经 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断或染色体易位。 这是导致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。二 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。Dr。 马蒂·盖勒特在NIH的团队是第一个展示 RAG-1和RAG-2蛋白在体外可启动基因重排。 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。在制作完 几十种不同的融合结构，我们终于成功地 在大肠杆菌中制造有活性的RAG-1，这为两种突变铺平了道路 对这一极其重要的物质的研究以及结晶学研究 蛋白。我们还克隆了人类RAG蛋白，并构建了 在大肠杆菌和昆虫细胞中均有表达。最终，我们将会 确定RAG蛋白的三维结构及其相互作用 X-射线与DNA识别序列的络合物 结晶学技术基因必须在每一次 细胞分裂的周期。尽管DNA聚合酶有一个校对 偶尔可将复制过程中的错误降至最低的机制 由于复制而导致的不匹配-仍会发生错误。在所有的生命中 生物体中有错配修复系统来防止这种突变 阻止它的发生。大肠杆菌有一个甲基导向的错配修复系统 由MutS、MutL和Muth蛋白组成。MutS和MutL的同系物 在人类体内也发现了蛋白质。这些蛋白质的突变是 90%的遗传性非息肉病性结直肠癌的罪魁祸首。 在去年，我们已经生长了Muth的晶体，一种特定于序列的 被MutS识别后被激活的内切酶 不匹配。我们得到了两种晶型的MuTH，都是衍射型的 从X射线到原子分辨率。最近我们解决并提炼了 MUTH，一条229aa的单一多肽链，在三个氨基酸中的结构 不同的水晶包装环境。我们还获得了晶体 目前正在解决其结构问题。

V(D)J gene rearrangement in vertebrates is essential for the maturation of immune systems. It allow 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 which are causes for 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 turn on the gene rearrangement in vivo. Dr. Marty Gellert's group here 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 making dozens of different fusion constructs, we have finally succeeded in making active RAG-1 in E. coli, which paves the road for both mutational studies as well as crystallographic studies of this extremely important protein. 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-dimensional structures of RAG proteins and their complexes with the DNA recognition sequences using x-ray crystallographic techniques. 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. The homologues of MutS and MutL proteins are also found in human. Mutations in these proteins are culprits in 90% of the hereditary nonpolyposis colorectal cancers. During last year, we have grown crystals of MutH, a sequence-specific endonuclease which is activated by MutS upon its recognition of mismatch. We have obtained two crystal forms of MutH, both diffract x-ray to atomic resolution. Recently we have solved and refined the structures of MutH, a single polypeptide chain of 229 aa, in three different crystal packing environments. We have also obtained crystals of MutL and are currently in the process of solving its structure.