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

在本项目中，我们旨在阐明大肠杆菌Ruva、RuvB和RuvC蛋白对Holliday连接解决素的分子机制，并寻找参与真核细胞重组中间产物解析的基因。我们的主要成果如下：1.我们用X射线结晶学和电子显微镜研究了Ruva-RuvB蛋白复合体的三维结构，并提出了Holliday结DNA与RuvAB蛋白的复合体模型。我们鉴定了10个与裂殖酵母重组修复相关的新基因，并对它们的功能进行了分析。通过单分子成像分析，研究了RuvAB复合体对Holliday连接的分支迁移机制，表明分支迁移是由DNA双链在连接处的旋转引起的。其中一个新发现的基因FBH-1编码一种带有DNA解旋酶和F-box基序的蛋白质。FBH-1蛋白参与了某种形式的重组中间产物的加工。我们研究了rad60和rad62(Nse4)基因的功能，发现它们在功能和物理上都与Smc5/6(染色体结构维护)复合体相关，并参与重组中间产物的加工。我们阐明了Rad60和Smc5/6复合体在检查点调控中的作用。我们在分裂酵母中发现了RAD51-Swi5-Sfr1蛋白复合体的一条新的重组修复途径。我们在嗜热古生菌中发现了一种结构特异的DNA解旋酶/核酸酶，命名为Hef-1，并阐明了其晶体结构。最近的一项发现突显了这种酶在DNA修复中的重要性，即它是范科尼贫血中突变的一个基因的同源基因。范科尼贫血是一种遗传性疾病，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.