Crystallization and preliminary diffraction analysis of the HincII restriction endonuclease-DNA complex
ABSTRACT Crystals of the 60 kDa dimeric HincII restriction enzyme bound to a 12 base-pair dyad-symmetric duplex DNA carrying the specific 5'-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%.
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ABSTRACT: The crystal structure of the HincII restriction endonuclease-DNA complex shows that degenerate specificity for blunt-ended cleavage at GTPyPuAC sequences arises from indirect readout of conformational preferences at the center pyrimidine-purine step. Protein-induced distortion of the DNA is accomplished by intercalation of glutamine side chains into the major groove on either side of the recognition site, generating bending by either tilt or roll at three distinct loci. The intercalated side chains propagate a concerted shift of all six target-site base pairs toward the minor groove, producing an unusual cross-strand purine stacking at the center pyrimidine-purine step. Comparison of the HincII and EcoRV cocrystal structures suggests that sequence-dependent differences in base-stacking free energies are a crucial underlying factor mediating protein recognition by indirect readout.Nature Structural Biology 02/2002; 9(1):42-7. DOI:10.1038/nsb741
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ABSTRACT: Repressor activator protein 1 (Rap1) is an essential factor involved in transcription and telomere stability in the budding yeast Saccharomyces cerevisiae. Its interaction with DNA causes hypersensitivity to potassium permanganate, suggesting local DNA melting and/or distortion. In this study, various Rap1-DNA crystal forms were obtained using specifically designed crystal screens. Analysis of the DNA conformation showed that its distortion was not sufficient to explain the permanganate reactivity. However, anomalous data collected at the Mn edge using a Rap1-DNA crystal soaked in potassium permanganate solution indicated that the DNA conformation in the crystal was compatible with interaction with permanganate ions. Sequence-conservation analysis revealed that double-Myb-containing Rap1 proteins all carry a fully conserved Arg580 at a position that may favour interaction with permanganate ions, although it is not involved in the hypersensitive cytosine distortion. Permanganate reactivity assays with wild-type Rap1 and the Rap1[R580A] mutant demonstrated that Arg580 is essential for hypersensitivity. AFM experiments showed that wild-type Rap1 and the Rap1[R580A] mutant interact with DNA over 16 successive binding sites, leading to local DNA stiffening but not to accumulation of the observed local distortion. Therefore, Rap1 may cause permanganate hypersensitivity of DNA by forming a pocket between the reactive cytosine and Arg580, driving the permanganate ion towards the C5-C6 bond of the cytosine.Acta Crystallographica Section D Biological Crystallography 03/2013; 69(Pt 3):409-19. DOI:10.1107/S0907444912049311 · 7.23 Impact Factor
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ABSTRACT: 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.Journal of Molecular Biology 09/2008; 383(1):186-204. DOI:10.1016/j.jmb.2008.08.032 · 3.96 Impact Factor