Structural Studies Of DNA Recombination And Repair

基因组DNA必须在每个细胞分裂周期之前进行复制。尽管复制性DNA聚合酶具有内建的校对机制，以尽量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。大多数生物体中都存在防止这种突变发生的错配修复系统。大肠杆菌具有甲基定向错配修复系统，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。在90%的遗传性非息肉病性结直肠癌中发现了这些蛋白的突变。在2000年10月之前的三年中，我们小组确定了错配修复蛋白MutH的晶体结构，MutL的保守的40KD ATPase片段及其核苷酸复合物，以及单独与DNA络合的190 Kd Taq MutS，以及与DNA和ADP/Mg2+的三元络合物。去年（2000-2001），我们进行了大肠杆菌MutS的诱变和生化研究，并基于我们的研究结果提出了atp辅助错配识别校对机制，这类似于蛋白质合成中的校对；(2)我们确定了MutL的人类同源物Pms2的atp酶片段的晶体结构，并表征了其不寻常的单分子atp酶活性；(3)与Thomas Kunkel博士合作。我们发起了异二聚体真核MutS同源物的不对称DNA结合和atp酶水解的研究，并表征了错配识别所需的残基与一般蛋白质-DNA相互作用所需的残基。(4)基于我们的晶体结构，我们在大肠杆菌MutS， MutL和MutH中产生了47个功能突变，并与加州大学洛杉矶分校的Jeffrey Miller博士合作，我们研究了它们的体内错配修复和DNA重组表型。结合我们的体外研究，我们已经产生了这些错配修复蛋白的功能和机制的全面概况。最近发现了一个新的DNA聚合酶家族——y家族。它们不同于先前已知的DNA聚合酶在初级序列和在病变绕过和易出错的DNA合成。在与NICHD的Roger Woodgate博士的合作下，我们确定了来自S. Solfotaricus的y家族DNA聚合酶Dpo4与底物DNA和进入的核苷酸的复合物的晶体结构。晶体结构提供了作用中的y家族聚合酶的第一个视图，并揭示了低保真DNA合成和绕过修饰DNA碱基的分子机制。我们继续进行V(D)J复合的研究。V(D)J基因在脊椎动物中的重排对免疫系统的成熟至关重要，这使得抗体和t细胞受体的产生能够建立防御系统。V(D)J基因重排是一种位点特异性DNA重组。ag -1和ag -2（重组激活基因产物）这两种蛋白在体内是开启基因重排的必要和充分的，但它们很难以纯的和可溶的形式获得。我们已经克隆并生产了几个小鼠RAG1蛋白的可溶性片段。尽管这些碎片是可溶的、纯净的、供应充足的，但它们仍然抵抗我们的结晶尝试。有趣的是，其中一个片段含有核酸酶活性，可能具有细胞功能。我们与麻省总医院的Marjorie Oettinger博士合作，对核酸酶的活性进行了表征。

Genomic DNA has to be replicated before every cycle of cell division. Although replicative DNA polymerase has a built-in proofreading mechanism to minimize the errors during replication, occasionally mismatch due to replication-errors still happens. Mismatch repair systems to prevent such mutations from occurring exist in most organisms. 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 nonpolyposis colorectal cancers. In the three years before Oct. 2000, our group determined the crystal structures of mismatch repair proteins MutH, a conserved 40KD ATPase fragment of MutL and its complexes with nucleotides, and the 190 Kd Taq MutS alone, complexed with DNA, and as a ternary complex with DNA and ADP/Mg2+. Last year (2000-2001), (1) we carried out mutagenesis and biochemical studies of E. coli MutS and based on our results proposed an ATP-assisted mismatch recognition proofreading mechanism, which is similar to the proofreading in protein synthesis, (2) we determined the crystal structure of the ATPase fragment of Pms2, a human homolog of MutL and characterized its unusual monomeric ATPase activity, (3) in collaboration with Dr. Thomas Kunkel, we initiated the studies of asymmetric DNA binding and ATPase hydrolysis in heterodimeric eukaryotic MutS homologs and characterized the residues essential for mismatch recognition versus residues required for general protein-DNA interactions, (4) we generated 47 functional mutations in E. coli MutS, MutL and MutH based on our crystal structures and in collaboration with Dr. Jeffrey Miller of UCLA, we studied their in vivo mismatch repair and DNA recombination phenotypes. Combined with our in vitro studies, we have produced a comprehensive profile of the function and mechanism of these mismatch repair proteins. A new family of DNA polymerases, the Y-family, has recently been identified. They differ from the previously known DNA polymerase in the primary sequence and in lesion-bypass and error-prone DNA synthesis. In collaboration with Dr. Roger Woodgate of NICHD, we have determined the crystal structure of a Y-family DNA polymerase, Dpo4 from S. Solfotaricus, in complex with the substrate DNA and an incoming nucleotide. The crystal structures provide the first view of the Y-family polymerase in action and reveal the molecular mechanism for low fidelity DNA synthesis and bypassing modified DNA bases. We have continued our research of V(D)J recombination. V(D)J gene rearrangement in vertebrates is essential for the maturation of immune systems, which allows the generation of antibodies and T-cell receptors to build up the defense system. 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, but they are difficult to obtain in a pure and soluble form. We have cloned and produced several soluble fragments of the mouse RAG1 proteins. Even though soluble, pure and in ample supply, these fragments still resist our crystallization attempts. Interestingly, one of the fragments contains a nuclease activity, which might have cellular functions. In collaboration with Dr. Marjorie Oettinger of Massachusetts General Hospital, we are characterizing the nuclease activity.