Restriction endonuclease BpuJI specific for the 5'-CCCGT sequence is related to the archaeal Holliday junction resolvase family.
Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5'-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3 nt from the 3'-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.
Sukackaite
Rasa
R
Institute of Biotechnology, Graiciūno 8, Vilnius, Lithuania.
Lagunavicius
Arunas
A
Stankevicius
Kornelijus
K
Urbanke
Claus
C
Venclovas
Ceslovas
C
Siksnys
Virginijus
V
D019843:Archaeal Proteins / Q000145:classification; D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000145:classification* / Q000378:metabolism; D019281:Dimerization; D045565:Holliday Junction Resolvases / Q000145:classification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D019843:Archaeal Proteins; D004247:DNA; D045565:Holliday Junction Resolvases; D015252:Deoxyribonucleases, Type II Site-Specific