The three-dimensional X-ray crystal structure of the ‘rare cutting’ type II restriction endonuclease SgrAI bound to cognate DNA is presented. SgrAI forms a dimer bound to one duplex of DNA. Two Ca2+ bind in the enzyme active site, with one ion at the interface between the protein and DNA, and the second bound distal from the DNA. These sites are differentially occupied by Mn2+, with strong binding at the protein–DNA interface, but only partial occupancy of the distal site. The DNA remains uncleaved in the structures from crystals grown in the presence of either divalent cation. The structure of the dimer of SgrAI is similar to those of Cfr10I, Bse634I and NgoMIV, however no tetrameric structure of SgrAI is observed. DNA contacts to the central CCGG base pairs of the SgrAI canonical target sequence (CR|CCGGYG, | marks the site of cleavage) are found to be very similar to those in the NgoMIV/DNA structure (target sequence G|CCGGC). Specificity at the degenerate YR base pairs of the SgrAI sequence may occur via indirect readout using DNA distortion. Recognition of the outer GC base pairs occurs through a single contact to the G from an arginine side chain located in a region unique to SgrAI.

SummaryFive new structures of the Q138F HincII enzyme bound to a total of three different DNA sequences and three different metal ions (Ca 2+ , Mg 2+ , and Mn 2+ ) are presented. While previous structures were produced from soaking Ca 2+ into pre-formed Q138F HincII/DNA crystals, the new structures are derived from cocrystallization with Ca 2+ , Mg 2+ , or Mn 2+ . The Mn 2+ bound structure provides the first view of a product complex of Q138F HincII with cleaved DNA. Binding studies and a crystal structure show how Ca 2+ allows trapping of a Q138F HincII complex with noncognate DNA in a catalytically incompetent conformation. Many Q138F HincII/DNA structures show asymmetry, despite the binding of a symmetric substrate by a symmetric enzyme. The various complexes are fit into a model describing the different conformations of the DNA bound enzyme, and show how DNA conformational energetics determine DNA cleavage rates by the Q138F HincII enzyme.Many fundamentally important functions within cells are mediated by proteins which recognize and bind to specific DNA sequences. Studies of sequence specific DNA binding proteins indicate that specific target sequences are recognized using both direct and indirect readout 1 . Direct readout involves direct contacts such as hydrogen bonds and van der Waals interactions between the protein and the DNA base functional groups, mostly in the major groove. Indirect readout is defined as sequence specificity occurring in the absence of such direct contacts, and has been proposed to involve contacts mediated by water 2 or other small molecules 3 , as well as distortions of DNA that can distinguish different sequences energetically 4-13 (Little, et al., submitted for publication).Restriction endonucleases are a class of enzymes found in bacteria which cleave DNA at specific target sequences and are thought to protect the bacterial cell from phage infection 14 . The target sites in the host DNA are methylated by a methyltransferase, and thereby protected from the cleavage activity of the endonuclease. Cleavage at sequences other than the target site can result in damage and possibly death to the bacterial cell, as only the target site sequence is protected by the activity of the cognate methyltransferase enzyme. Therefore, restriction *Corresponding author, NCH: Telephone: 520-626-3828 FAX: 520-626-9288 nhorton@u.arizona.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCESSION NUMBERSCoordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 3E3Y, 3E40, 3E41, 3E42, 3E43, 3E44, 3E45. NIH Public...

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