The 2.8 A crystal structure of the type II restriction endonuclease HincII bound to Ca(2+) and cognate DNA containing GTCGAC is presented. The DNA is uncleaved, and one calcium ion is bound per active site, in a position previously described as site I in the related blunt cutting type II restriction endonuclease EcoRV [Horton, N. C., Newberry, K. J., and Perona, J. J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95 (23), 13489-13494], as well as that found in other related enzymes. Unlike the site I metal in EcoRV, but similar to that of PvuII, NgoMIV, BamHI, BglII, and BglI, the observed calcium cation is directly ligated to the pro-S(p) oxygen of the scissile phosphate. A calcium ion-ligated water molecule is well positioned to act as the nucleophile in the phosphodiester bond cleavage reaction, and is within hydrogen bonding distance of the conserved active site lysine (Lys 129), as well as the pro-R(p) oxygen of the phosphate group 3' of the scissile phosphate, suggesting possible roles for these groups in the catalytic mechanism. Kinetic data consistent with an important role for the 3'-phosphate group in DNA cleavage by HincII are presented. The previously observed sodium ion [Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47] persists in the active sites of the Ca(2+)-bound structure; however, kinetic data show little effect on the single-turnover rate of DNA cleavage in the absence of Na(+) ions.

High resolution x-ray crystal structures of protein-DNA complexes have revealed a rational basis for sequence specificity in the form of direct contacts between amino acid side chains and DNA bases in the major groove (1, 2). However, cases have been reported where specificity of a protein for a particular base pair (or base pairs) within its recognition sequence exists even when no direct contacts are seen between the protein and DNA (3-11). In these cases the specificity exhibited by the DNA-binding protein appears to be manifested by indirect means. This "indirect readout" of the DNA sequence may involve water-mediated contacts, contacts to the sugar-phosphate backbone, or the utilization of sequence-dependent DNA structure and/or deformability (3, 12).The crystal structure of the type II restriction endonuclease HincII reveals distortion from canonical B-form of its bound cognate DNA (13). HincII recognizes and cleaves the sequence GTYRAC, having degeneracy in its specificity for the center two nucleotides by allowing the presence of any pyrimidinepurine, i.e. TA, TG, CA, or CG. Sequence-specific contacts between the protein and DNA can explain this specificity. The contact to the center YR step (CG in the original crystal structure (13)) consists of a hydrogen bond from Asn-141 to the N7 of the purine base. Such a contact can occur when either G or A, but not C or T, occur in the fourth position of the recognition sequence. No contacts are made to the pyrimidine base. Typically, the high degree of specificity achieved by type II restriction endonucleases is due in part to the fact that contacts occur to both bases of each base pair. The question remains of whether or not one hydrogen bond to each purine of the center base pairs can explain the HincII specificity against sequences containing GTYYAC, GTRYAC, or GTRRAC.The crystal structure of wild type HincII bound to one of the three cognate DNA sequences, GTCGAC, shows that HincII distorts the bound DNA at three loci, two as a result of intercalation of the Gln-138 side chain into the DNA duplex just outside of its recognition sequence (Fig. 1A, light brown spacefilling atoms ϭ the recognition sequence, dark brown spacefilling atoms ϭ flanking DNA), and the third site at the center YR base pair (Fig. 1, A, red ϭ Y (C or T), and blue ϭ R (A or G), B, right). A hypothesis has been put forth that links the distortion of DNA at the center CG to indirect readout of this sequence by HincII. Furthermore, modeling of the DNA distortion revealed a connection between the Gln-138 intercalation and the center base pair distortion three base pairs away (13).The studies herein describe the characterization of HincII structure and function when glutamine 138 has been substituted with phenylalanine, Q138F. Preliminary velocity measurements showed that Q138F HincII favors TA over CG at the center YR step to a greater extent than wild type. Subsequent DNA binding assays and single turnover cleavage rate measurements revealed that the alteration in specificity toward cognate ...

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Crystals of a designed six-finger zinc-finger protein, Aart, bound to a 22-basepair duplex DNA containing a consensus binding site have been obtained. Crystals grew by hanging-drop vapor diffusion from solutions containing polyethylene glycol 4000 as the precipitating agent. The irregularly shaped crystals belong to space group P1, with unit-cell parameters a = 41.95, b = 71.76, c = 74.73 Å , = 100.87, = 96.22, = 106.33 . There are most likely to be two protein-DNA complexes in the asymmetric unit. A complete native data set has been collected from a high-energy synchrotron source to a resolution of 2.95 Å at 100 K, with an R merge of 9.3%.

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