Acta Cryst. (1999). D55, 1943–1945 Horton et al.  HincII –DNA complex 1943 crystallization papers Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Crystallization and preliminary diffraction analysis of the HincII restriction endonuclease–DNA complex Nancy C. Horton, a Lydia F. Dorner, b Ira Schildkraut b and John J. Perona a * a Department of Chemistry and Interdepartmental Program in Biochemistry and Molecular Biology, University of California at Santa Barbara, Santa Barbara CA 93106-9510, USA, and b New England Biolabs, 32 Tozer Road, Beverly, MA 01915, USA Correspondence e-mail: perona@chem.ucsb.edu # 1999 International Union of Crystallography Printed in Denmark – all rights reserved Crystals of the 60 kDa dimeric HincII restriction enzyme bound to a 12 base-pair dyad-symmetric duplex DNA carrying the specific 5 0 -GTCGAC recognition site have been obtained. Crystals grew by hanging-drop vapor diffusion from solutions containing polyethylene glycol 4000 as precipitating agent. The rod-shaped crystals belong to space group I222 (or I2 1 2 1 2 1 ), with unit-cell dimensions a = 66.9, b = 176.7, c = 256.0 A ˚ . There are most likely to be two dimeric complexes in the asymmetric unit. A complete native data set has been collected from a high-energy synchrotron source to a resolution of 2.5 A ˚ at 100 K, with an R merge of 4.8%. Received 25 May 1999 Accepted 2 August 1999 1. Introduction Type II restriction endonucleases are superb models for the study of protein–DNA inter- actions because of their exceptionally high specificities for cleavage of target DNA sites (Roberts & Halford, 1993). Typically, mutation of a single nucleotide of a 4–8 base-pair dyad- symmetric recognition sequence decreases rate constants by 10 5 -fold to 10 6 -fold (Lesser et al., 1990), as required for function in a bacterial host defensive system. Crystal structures of six type II restriction endonucleases have been determined: EcoRI (Rosenberg, 1991), EcoRV (Winkler et al., 1993), PvuII (Athanasiadis et al., 1994; Cheng et al., 1994), BamHI (Newman et al., 1995), Cfr10I (Bozic et al., 1996) and BglI (Newman et al., 1998). The type IIs enzyme FokI has also been solved (Wah et al., 1997). All of the structures except Cfr10I are deter- mined bound to DNA. Little primary sequence similarity exists among the enzymes; thus, the use of sequence information to help under- stand structure–function relationships has been limited. The four enzymes EcoRI, BamHI, EcoRV and PvuII are divisible into those which cleave leaving four-base 5 0 -overhangs (EcoRI, BamHI) and those which cleave at the center step to produce blunt-ended products (EcoRV, PvuII). While topologically similar folds char- acterize each subclass (Aggarwal, 1995), large differences in structure still remain. For example, comparison of EcoRV and PvuII shows that while three central -strands are superimposable with an r.m.s. deviation of 0.8 A ˚ , flanking elements in the conserved active-site domain deviate substantially. The dimer interfaces of the two enzymes are completely unrelated. Most significantly, even the general modes of DNA recognition are not the same: PvuII binds unbent B-form DNA using antiparallel -structure, whereas EcoRV employs surface loops and bends the DNA sharply into the major groove. The small number of known type II restric- tion-enzyme structures poses severe limita- tions on our ability to rationally engineer new sequence specificities. It is already known that the introduction of small numbers of amino- acid substitutions in the DNA-binding clefts of EcoRI and EcoRV does not result in specificity modification, but rather in reduced activities toward both wild-type and modified target sites (Heitman & Model, 1990; Flores et al. , 1995; Wenz et al., 1994; Lanio et al. , 1996). An alternative approach toward understanding and ultimately manipulating sequence prefer- ences is to generate a database of structures within a subclass where there is precedent for topological similarity within the context of different specificities. Comparisons among such enzymes should lead to an improved understanding of the roles of individual poly- peptide regions and amino acids in generating the extraordinary DNA-sequence selectivities. Here, we report crystals of restriction endonuclease HincII bound to a duplex DNA molecule containing the specific recognition site. HincII recognizes the sequence 5 0 -GTPyPuAC and cleaves it in a blunt-ended fashion at the center Py–Pu step (Kelly & Smith, 1970). It is thus identical with the blunt- cutting EcoRV (5 0 -GATATC) at the outer pair, with similar but degenerate specificity at the inner base-pair step. Interestingly, a limited sequence similarity of HincII with EcoRV in the region of the major-groove binding recog- nition loops (R-loops) of EcoRV has been noted (Thielking et al., 1991). This similarity provides some further confidence that the HincII and EcoRV active-site domains will be topologically similar. It is known that EcoRV utilizes the sequence-dependent free-energy