3bm3

Restriction endonuclease PspGI-substrate DNA complex

spinqualitylabelsall models 3bm3, resolution 1.70Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3bm3 is a 4 chain structure with sequence from Pyrococcus sp. gi-h. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: CIT NonStd Res: MSE Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI-DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution. Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence.,Tamulaitis G, Zaremba M, Szczepanowski RH, Bochtler M, Siksnys V Nucleic Acids Res. 2007;35(14):4792-9. Epub 2007 Jul 7. PMID:17617640 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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