Crystal structure of the -Me type II restriction endonuclease Hpy99I with target DNA

The -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 -barrel and two 42 repeats. Each repeat coordinates a structural zinc ion with four cysteine thiolates in two CXXC motifs. The -Me region of the second 42 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 42 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 Hpy99IDNA co-crystal structure provides the first detailed illustration of the -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).