王惠鈞Wang, Andrew H.-J.臺灣大學:生化科學研究所張育瑋2010-05-042018-07-062010-05-042018-07-062008U0001-2407200815335200http://ntur.lib.ntu.edu.tw//handle/246246/178844Homologous recombination is a universal mechanism for repairing DNA double strand breaks (DSBs) and injured DNA replication forks. RecA family proteins play a central role in homologous recombination by forming nucleoprotein filaments. These proteins include the prokaryotic RecA, archaeal RadA, and eukaryotic Rad51 and Dmc1. Here we report three crystal structures of the Sulfolobus solfataricus RadA. All of these structures are packed in the orthorhombic lattice and form the left-handed helical filaments with different helical pitches. The results strongly suggest the universal existence of left-handed helical filaments of the RecA family proteins. The structural variations of alternate protein-protein interfaces not only exhibit the extreme flexibility of filaments but also demonstrate the asymmetry within the filaments, implying the sequential binding of DNA and hydrolysis of ATP by RecA family proteins. Further structural analyses and subsequent mutagenesis studies, coupled with biochemical assays, offer a novel insight into the mechanism controlling the SsoRadA quaternary structures and filament assemblies during the catalysis of strand exchange, which involves Asp 70 and Arg 72 in the N-terminus of the NTD-CTD hinge.誌謝 i文摘要 iiBSTRACT iiiONTENTS ivIST OF FIGURES viIST OF TABLES viiNTRODUCTION 1ATERIALS AND METHODS .............................................................. 6xpression and Purification of SsoRadA 6rystallization, Data Collection and Structure Determination of SsoRadA 7NA Substrates 8NA Binding Assay 9sDNA-stimulated ATPase Activity Assays 9trand Assimilation Assays 10ESULTS 11verall Structures of SsoRadA 0522, 0719, and 1007 11he Asymmetry of SsoRadA Protein Filaments 12he N-terminus of the NTD-CTD Hinge Accounts for Different Orientation of NTDs 14nalyses of Point Mutations Indicate that SsoRadA Asp70 and Arg72 Play Crucial Roles in Strand Paring Reaction 16ISCUSSION 20he Orthorhombic Crystals of Left-handed RecA Family Protein Filaments 20mplication by the Asymmetry in the Filaments 21he Effects of DNA on SsoRadA 22tructural Analysis Combining Biochemical Assays Indicates that D70 and R72 are Involved in Controlling the Quaternary Structures of SsoRadA Filaments 24he Limitation of the Resolution of RadA/Rad51 Crystal Structures 27ONCLUSION 28IGURES 30ABLES 48EFERENCES 52OSTER 60PPENDIX 62application/pdf8383486 bytesapplication/pdfen-US同源重組RECARADA斜方晶系晶體結構左手旋蛋白質絲狀聚合體蛋白質絲狀聚合體的組合Homologous recombinationRecA family proteinsRadAorthorhombic crystal structuresleft-handed protein filamentsfilament assemblyhttp://ntur.lib.ntu.edu.tw/bitstream/246246/178844/1/ntu-97-R95b46010-1.pdf