Mechanisms of Formation and Resolution of Holliday junction in Recombination and Replication

本课题旨在阐明E.大肠杆菌RuvA，RuvB和RuvC蛋白，并发现参与真核生物中重组中间体的分解的基因。我们的主要成果如下：1.通过X射线晶体学和电子显微镜研究了RuvA-RuvB蛋白复合物的三维结构，提出了RuvAB蛋白复合物与Holliday连接DNA的模型.我们在裂殖酵母中鉴定了10个参与重组修复的新基因，并分析了它们的功能.通过单分子图像分析研究了RuvAB复合物引起Holliday连接处分支迁移的机制，发现分支迁移是由连接处DNA双链体的旋转引起的.其中一个新发现的基因fbh-1编码一种具有DNA解旋酶和F-box基序的蛋白质。Fbh-1蛋白参与加工某种形式的重组中间体.我们研究了rad 60和rad 62（nse 4）基因的功能，发现它们与Smc 5/6（Structural Maintenance of Chromosome）复合体在功能和物理上都有联系，并参与重组中间体的加工。我们阐明了Rad 60和Smc 5/6复合物在检查点调节中的作用。我们发现了一个新的重组修复途径的Rad 51-Swi 5-Sfrl蛋白复合物在分裂酵母中.我们在嗜热古菌中发现了一种结构特异的DNA解旋酶/核酸酶Hef-1，并对其晶体结构进行了鉴定。最近的发现强调了这种酶在DNA修复中的重要性，即它是范可尼贫血症中突变基因之一的直系同源物，范可尼贫血症是一种遗传性疾病，具有DNA修复缺陷。

In this project, we aimed at elucidating molecular mechanisms of Holliday junction resolutin by E. coli RuvA, RuvB, and RuvC proteins, and finding genes involved in resolution of recombination intermediates in eukaryotes. Our major achievements are as follows.1. We elucidated three-dimensional structure of RuvA-RuvB protein complex by X-ray crystallography and electron microscopy, and proposed a model of Holliday junction DNA complexed with RuvAB proteins.2. We identified 10 novel genes involved, in recombination repair in fission yeast and analyzed their functions.3. We studied the mechanism of branch migration of Holliday junction by RuvAB complex by single-molecule imaging analysis and showed that branch migration is brought by rotation of DNA duplex at the junction.4. One of the newly found gene, fbh-1, encodes a protein with DNA helicase and F-box motifs. Fbh-1 protein is involved in processing some form of recombination intermediate.5. We studied the functions of rad60 and rad62 (nse4) genes and found that they are functionally and physically associate with Smc5/6 (Structural Maintenance of Chromosome) complex, and are involved in processing recombination intermediates. We elucidated role of the Rad60 and Smc5/6 complex in checkpoint regulation.6. We discovered a new recombination repair pathway by the Rad51-Swi5-Sfrl protein complex in fission yeast.7. We discovered a structure-specific DNA helicase/nuclease named Hef-1 in thermophilic archaea and elucidated the crystal structure. The importance of this enzyme in DNA repair is highlighted by the recent finding that it is an ortholog of one of the genes mutated in Fanconi Anemia, a hereditary disease having a defect in DNA repair.