Domain swapping in allosteric modulation of DNA specificity.

SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of cleavage activity and sequence specificity. Previous studies have shown that DNA bound dimers of SgrAI oligomerize into an activated form with higher DNA cleavage rates, although previously determined crystal structures of SgrAI bound to DNA show only the DNA bound dimer. A new crystal structure of the type II restriction endonuclease SgrAI bound to DNA and Ca(2+) is now presented, which shows the close association of two DNA bound SgrAI dimers. This tetrameric form is unlike those of the homologous enzymes Cfr10I and NgoMIV and is formed by the swapping of the amino-terminal 24 amino acid residues. Two mutations predicted to destabilize the swapped form of SgrAI, P27W and P27G, have been made and shown to eliminate both the oligomerization of the DNA bound SgrAI dimers as well as the allosteric stimulation of DNA cleavage by SgrAI. A mechanism involving domain swapping is proposed to explain the unusual allosteric properties of SgrAI via association of the domain swapped tetramer of SgrAI bound to DNA into higher order oligomers.

New clues in the allosteric activation of DNA cleavage by SgrAI: structures of SgrAI bound to cleaved primary-site DNA and uncleaved secondary-site DNA.

SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-activation with expansion of DNA-sequence specificity. The three-dimensional crystal structures of SgrAI bound to cleaved primary-site DNA and Mg²(+) and bound to secondary-site DNA with either Mg²(+) or Ca²(+) are presented. All three structures show a conformation of enzyme and DNA similar to the previously determined dimeric structure of SgrAI bound to uncleaved primary-site DNA and Ca²(+) [Dunten et al. (2008), Nucleic Acids Res. 36, 5405-5416], with the exception of the cleaved bond and a slight shifting of the DNA in the SgrAI/cleaved primary-site DNA/Mg²(+) structure. In addition, a new metal ion binding site is located in one of the two active sites in this structure, which is consistent with proposals for the existence of a metal-ion site near the 3'-O leaving group.

The structure of SgrAI bound to DNA; recognition of an 8 base pair target.

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 Ca(2+) 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 Mn(2+), 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.

Early interrogation and recognition of DNA sequence by indirect readout.

Control of replication, transcription, recombination and repair requires proteins capable of finding particular DNA sequences in a background of a large excess of nonspecific sequences. Such recognition can involve direct readout, with direct contacts to the bases of DNA, or in some cases through the less well-characterized indirect readout mechanisms. In order to measure the relative contributions of direct and indirect readout by a sequence specific endonuclease, HincII, a mutant enzyme deficient in a direct contact, was characterized, and surprisingly showed no loss of sequence specificity. The three dimensional crystal structure shows the loss of most of the direct readout contacts to the DNA, possibly capturing an early stage in target site recognition using predominately indirect readout to prescreen sites before full sequence interrogation.

The restriction enzyme SgrAI: structure solution via combination of poor MIRAS and MR phases.

Uninterpretable electron-density maps were obtained using either MIRAS phases or MR phases in attempts to determine the structure of the type II restriction endonuclease SgrAI bound to DNA. While neither solution strategy was particularly promising (map correlation coefficients of 0.29 and 0.22 with the final model, respectively, for the MIRAS and MR phases and Phaser Z scores of 4.0 and 4.3 for the rotation and translation searches), phase combination followed by density modification gave a readily interpretable map. MR with a distantly related model located a dimer in the asymmetric unit and provided the correct transformation to use in averaging electron density between SgrAI subunits. MIRAS data sets with low substitution and MR solutions from only distantly related models should not be ignored, as poor-quality starting phases can be significantly improved. The bootstrapping strategy employed to improve the initial MIRAS phases is described.

DNA-induced conformational changes in type II restriction endonucleases: the structure of unliganded HincII.

The 2.1A crystal structure of the unliganded type II restriction endonuclease, HincII, is described. Although the asymmetric unit contains only a single monomer, crystal lattice contacts bring two monomers together to form a dimer very similar to that found in the DNA bound form. Comparison with the published DNA bound structure reveals that the DNA binding pocket is expanded in the unliganded structure. Comparison of the unliganded and DNA liganded structures reveals a simple rotation of subunits by 11 degrees each, or 22 degrees total, to a more closed structure around the bound DNA. Comparison of this conformational change to that observed in the other type II restriction endonucleases where DNA bound and unliganded forms have both been characterized, shows considerable variation in the types of conformational changes that can occur. The conformational changes in three can be described by a simple rotation of subunits, while in two others both rotation and translation of subunits relative to one another occurs. In addition, the endonucleases having subunits that undergo the greatest amount of rotation upon DNA binding are found to be those that distort the bound DNA the least, suggesting that DNA bending may be less facile in dimers possessing greater flexibility.

DNA distortion and specificity in a sequence-specific endonuclease.

Five 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 preformed 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.