1t9j
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(New page: 200px<br /> <applet load="1t9j" size="450" color="white" frame="true" align="right" spinBox="true" caption="1t9j, resolution 2.00Å" /> '''I-CreI(Q47E)/DNA co...)
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Revision as of 10:37, 8 November 2007
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I-CreI(Q47E)/DNA complex
Overview
The LAGLIDADG homing endonucleases include free-standing homodimers, pseudosymmetric monomers, and related enzyme domains embedded within, inteins. DNA-bound structures of homodimeric I-CreI and monomeric I-SceI, indicate that three catalytic divalent metal ions are distributed across a, pair of overlapping active sites, with one shared metal participating in, both strand cleavage reactions. These structures differ in the precise, position and binding interactions of the metals. We have studied the metal, dependence for the I-CreI homodimer using site-directed mutagenesis of, active site residues and assays of binding affinity and cleavage activity., We have also reassessed the binding of a nonactivating metal ion (calcium), in the wild-type enzyme-substrate complex, and determined the DNA-bound, structure of two inactive enzyme mutants. The conclusion of these studies, is that the catalytic mechanism of symmetric LAGLIDADG homing, endonucleases, and probably many of their monomeric cousins, involves a, canonical two-metal mechanism in each of two active sites, which are, chemically and structurally tethered to one another by a shared metal ion., Failure to occupy the shared metal site, as observed in the presence of, calcium or when the metal-binding side chain from the LAGLIDADG motif (Asp, 20) is mutated to asparagine, prevents cleavage by the enzyme.
About this Structure
1T9J is a Single protein structure of sequence from Chlamydomonas reinhardtii. Full crystallographic information is available from OCA.
Reference
Metal-dependent DNA cleavage mechanism of the I-CreI LAGLIDADG homing endonuclease., Chevalier B, Sussman D, Otis C, Noel AJ, Turmel M, Lemieux C, Stephens K, Monnat RJ Jr, Stoddard BL, Biochemistry. 2004 Nov 9;43(44):14015-26. PMID:15518550
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