![]() ![]() Of the 8 bp in the enzyme's target site, the central two A:T base pairs (at positions ☑) are disrupted. ( B) The DNA is bent by ∼50°, resulting in widening of the minor groove around the position of the two scissile phosphates. the nuclease domain in each subunit) display much more limited motions that allow formation of atomic contacts to individual atoms in the DNA. ( A) The protein closure shown in Figure 3 is largely facilitated by a change in the bending of the C-terminal helix in each protein subunit. Inset: Similar domain-swapped helices from a specificity (S) subunit from a type I R/M system in Methanocaldoccus jannaschii (PDB ID 1YF2) that dictate the distance between target recognition domains (TRDs) in type I enzyme assemblages.Ĭonformational changes that accompany protein-DNA binding. ( B) Close-up of the domain-swapped C-terminal helical bundle, showing the array of aliphatic and aromatic residues that form the packed interface between the helical protein backbones. Note the large basic cleft that separates the two folded nuclease domains. basic) regions are blue, while negatively charged (i.e. On the right are electrostatic surface charge representations of the enzyme homodimer, shown in the same orientation as the ribbon diagram. The folded domains that extend below those two helices correspond to the α/β fold that comprises the core nuclease domain from the PD-(D/E)xK family. The primary interface between the protein subunits is a long domain-swapped C-terminal helix that forms an antiparallel bundled structure similar to a coiled-coil. ( A) On the left are ribbon diagrams of the SwaI homodimer (shown in two orientations), with the two protein subunits colored green and cyan, respectively. Structure of the unbound (apo-enzyme) form of SwaI. ‘Y’ and ‘R’ refer to any pyrimidine and purine base, respectively, which corresponds to promiscuous recognition at positions ☑ by HincII. ( B) Sequence and numbering of the DNA target sites recognized and cleaved by SwaI, HincII and EcoRV. The ‘X48’ notation in the HincII sequence correspond to a large unique insertion of additional residues, that are not shown, relative to all the other aligned sequences. The position and boundaries of secondary structural elements in SwaI are shown above the aligned sequences, and the position of the conserved active site residues are indicated with red arrows. The overall identity between SwaI and its nearest sequence relatives is 46–57% identity, while the identity between SwaI and HincII or EcoRV is 14% and 11%, respectively (corresponding to 32 and 25 conserved residues, respectively). ![]() ![]() Conserved residues are shown as colored bold residues. One additional significant sequence homologue (WP_066163638 REBASE Bsp13219ORF4205P) is not shown for clarity. ( A) Alignment of SwaI against four of its closest sequence homologues identified within the NCBI protein database (39), and against its two nearest structural homologues (HincII and EcoRV) identified within the RCSB protein structure database (40). This result is reminiscent of a more dramatic target deformation previously described for PacI, implying that long A:T-rich target sites might display structural or dynamic behaviors that play a significant role in endonuclease recognition and cleavage. Binding by SwaI induces an extreme bend in the target sequence accompanied by un-pairing and re-ordering of its central A:T base pairs. We compare these structures to two structurally similar ‘PD-D/ExK’ restriction endonucleases (EcoRV and HincII) that also generate blunt-ended products, and to a structurally distinct enzyme (the HNH endonuclease PacI) that also recognizes an 8-bp target site consisting solely of A:T base pairs. Here, we report three crystal structures of SwaI: unbound enzyme, a DNA-bound complex with calcium ions and a DNA-bound, fully cleaved complex with magnesium ions. R.SwaI, a Type IIP restriction endonuclease, recognizes a palindromic eight base pair (bp) symmetric sequence, 5΄-ATTTAAAT-3΄, and cleaves that target at its center to generate blunt-ended DNA fragments. ![]()
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