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

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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.