The recognition domain of the BpuJI restriction endonuclease in complex with cognate DNA at 1.3-A resolution |
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Authors: | Sukackaite Rasa Grazulis Saulius Bochtler Matthias Siksnys Virginijus |
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Affiliation: | 1 Institute of Biotechnology, Graiciuno 8, 02241 Vilnius, Lithuania 2 International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland 3 Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01309 Dresden, Germany 4 Schools of Chemistry and Biosciences, Cardiff University, Park Place, Cardiff CF10 3AT, Wales, UK |
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Abstract: | Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5′-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-Å resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein-DNA complexes are different. Recognition of the 5′-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix-turn-helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis. |
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Keywords: | HTH, helix-turn-helix wt, wild type wHTH, winged helix-turn-helix PEG, polyethylene glycol |
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