Nucleic Acids Research Advance Access originally published online on April 20, 2009
Nucleic Acids Research 2009 37(11):3799-3810; doi:10.1093/nar/gkp228
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Nucleic Acids Research, 2009, Vol. 37, No. 11 3799-3810
© 2009 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.
Structural Biology |
Crystal structure of the ββ
-Me type II restriction endonuclease Hpy99I with target DNA
1International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland, 2Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr, 108, 01309 Dresden, Germany and 3Schools of Chemistry and Biosciences, Main Building, Park Place, Cardiff University, Cardiff CF10 3AT, UK
*To whom correspondence should be addressed. Tel: +48 22 5970732 or +44 29 208 70625; Fax: +48 22 5970715; Email: mbochtler{at}iimcb.gov.pl or bochtlerm{at}cardiff.ac.uk
Received January 6, 2009. Revised March 25, 2009. Accepted March 25, 2009.
The ββ
-Me restriction endonuclease (REase) Hpy99I recognizes the CGWCG target sequence and cleaves it with unusual stagger (five nucleotide 5'-recessed ends). Here we present the crystal structure of the specific complex of the dimeric enzyme with DNA. The Hpy99I protomer consists of an antiparallel β-barrel and two β42 repeats. Each repeat coordinates a structural zinc ion with four cysteine thiolates in two CXXC motifs. The ββ-Me region of the second β42 repeat holds the catalytic metal ion (or its sodium surrogate) via Asp148 and Asn165 and activates a water molecule with the general base His149. In the specific complex, Hpy99I forms a ring-like structure around the DNA that contacts DNA bases on the major and minor groove sides via the first and second β42 repeats, respectively. Hpy99I interacts with the central base pair of the recognition sequence only on the minor groove side, where A:T resembles T:A and G:C is similar to C:G. The Hpy99I–DNA co-crystal structure provides the first detailed illustration of the ββ-Me site in REases and complements structural information on the use of this active site motif in other groups of endonucleases such as homing endonucleases (e.g. I-PpoI) and Holliday junction resolvases (e.g. T4 endonuclease VII).