Crystal structure of the beta beta alpha-Me type II restriction endonuclease Hpy99I with target DNA.
The beta beta alpha-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 beta-barrel and two beta 4 alpha 2 repeats. Each repeat coordinates a structural zinc ion with four cysteine thiolates in two CXXC motifs. The beta beta alpha-Me region of the second beta 4 alpha 2 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 beta 4 alpha 2 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 beta beta alpha-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).
Sokolowska
Monika
M
International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
Czapinska
Honorata
H
Bochtler
Matthias
M
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry
D008670:Metals; D021122:Protein Subunits; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific