title: Metal ion and DNA binding by single-chain PvuII endonuclease: lessons from the linker.
abstract: Understanding the roles of metal ions in restriction enzymes has been complicated by both the presence of two metal ions in many active sites and their homodimeric structure. Using a single-chain form of the wild-type restriction enzyme PvuII (scWT) in which subunits are fused with a short polypeptide linker (Simoncsits et al. in J. Mol. Biol. 309:89-97, 2001), we have characterized metal ion and DNA binding behavior in one subunit and examined the effects of the linker on dimer behavior. scWT exhibits heteronuclear single quantum coherence NMR spectra similar to those of native wild-type PvuII (WT). For scWT, isothermal titration calorimetry data fit to two Ca(II) sites per subunit with low-millimolar K                         (d)s. The variant scWT|E68A, in which metal ion binding in one subunit is abolished by mutation, also binds two Ca(II) ions in the WT subunit with low-millimolar K                         (d)s. When there are no added metal ions, DNA binding affinity for scWT is tenfold stronger than that of the native WT, but tenfold weaker at saturating Ca(II) concentration. In the presence of Ca(II), scWT|E68A binds target DNA similarly to scWT, indicating that high-affinity substrate binding can be carried energetically by one metal-ion-binding subunit. Global analysis of DNA binding data for scWT|E68A suggests that the metal-ion-dependent behaviors observed for WT are reflective of independent subunit behavior. This characterization provides an understanding of subunit contributions in a homodimeric context.
authors: Department of Chemistry and Biochemistry, The Center for Nanoscience, University of Missouri St. Louis, USA.
authors: Grigorios A
authors: GA
authors: Papadakos
authors: Cynthia M
authors: CM
authors: Dupureur
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding
chemical_list: D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D008670:Metals; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium; D002118:Calcium