| Abstract [eng] |
Due to their unique specificity, restriction endonucleases (REases) have gained widespread application as indispensable tools for in vitro manipulation and cloning of DNA. Delineation of the repertoire of the protein folds, providing three-dimensional portraits of the REases by crystallographic methods, should reveal how different folds are tailored to function as restriction enzymes. The major goal of this work was to explore the specificity-structure relationships within PD-(D/E)XK and phospholipase D superfamily enzymes using a combination of the crystallographic and biochemical methods. More specifically, we have focused on the Bse634I and EcoRII restriction enzymes belonging to the Cfr10I/NgoMIV/Bse634I branch of PD-(D/E)XK superfamily, and BfiI REase of PLD superfamily. Dissertation presents the crystal structure of the Bse634I REase mutant (R226A) complexed with two alternative target sites 5'-ACCGGT and 5'-GCCGGC, respectively. The analysis of the crystal structures revealed for the first time that the degenerate base pairs recognition by Bse634I is achieved through the combination of direct and indirect readout mechanisms. The crystal structures of the N- and C-terminal domains of EcoRII solved in the DNA bound form revealed different structural mechanisms used for the recognition of the same target sequence 5'-CCWGG. The first structural evidence has been provided that the C-terminal domain of EcoRII (EcoRII-C) flips out the central nucleotides A/T while interacting with its target site, enabling the EcoRII-C to use symmetric conserved structural elements for the recognition of the CCGG core. Furthermore, the crystal structure of the EcoRII N-terminal domain (EcoRII-N) provided the first glimpse into the B3 family domain in the DNA bound form. Finally, the crystal structure of the C-terminal DNA binding domain of BfiI (BfiI-C) bound to the target site 5'-ACTGGG enabled the first structural comparison of two B3-family domains (EcoRII-N and BfiI-C) in the DNA-bound form. |
