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Nucleic Acids Research Advance Access originally published online on March 28, 2007
Nucleic Acids Research 2007 35(7):2377-2389; doi:10.1093/nar/gkm164
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Nucleic Acids Research, 2007, Vol. 35, No. 7 2377-2389
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Restriction endonuclease BpuJI specific for the 5'-CCCGT sequence is related to the archaeal Holliday junction resolvase family

Rasa Sukackaite1, Arunas Lagunavicius1, Kornelijus Stankevicius1, Claus Urbanke2, Ceslovas Venclovas1 and Virginijus Siksnys1,*

1Institute of Biotechnology, Graiciuno 8, LT-02241 Vilnius, Lithuania and 2Strukturanalyse, Medizinische Hochschule Hannover, Carl Neuberg Strasse 1, D-30632 Hannover, Germany

*To whom correspondence should be addressed. Email: siksnys{at}ibt.lt

Received February 1, 2007. Revised February 15, 2007. Accepted March 5, 2007.

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.

Present address: Arunas Lagunavicius, Fermentas UAB, Graiciuno 8, LT-02241 Vilnius, Lithuania


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