| Abstract [eng] |
Type II restriction endonucleases recognize specific DNA sequences and cleave DNA at fixed positions within or close to this sequence. BpuJI recognizes the 5’-CCCGT sequence, but in contrast to other enzymes its cleavage site is very variable. This study shows that BpuJI is a dimer in solution and consists of two separate domains. The N-domain binds to the target sequence as a monomer, while the C-domain is responsible for nuclease activity and dimerization. The nuclease activity is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the N-domains. The activated C-domain cleaves DNA near the target site. In addition, it possesses an end-directed nuclease activity and preferentially cuts ~3 nt from the 3’ terminus. This leads to a very complicated pattern of DNA cleavage. Bioinformatics and mutational analysis revealed that the BpuJI C-domain harbours a PD (D/E)XK active site and is structurally related to archaeal Holliday junction resolvases. The crystal structure of the BpuJI N-domain bound to cognate DNA was solved at 1.3 Å resolution. It revealed two winged-helix subdomains, D1 and D2. The recognition of the target sequence is achieved the amino acid residues located on both the HTH motifs and an N-terminal arm. The BpuJI DNA recognition domain is most similar to the nicking endonuclease Nt.BspD6I. The modelling suggests that Nt.BspD6I could share the specificity-determining regions with BpuJI. |
