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| | ==I-LtrI E184D bound to non-cognate C4 substrate (pre-cleavage complex)== | | ==I-LtrI E184D bound to non-cognate C4 substrate (pre-cleavage complex)== |
| - | <StructureSection load='6bct' size='340' side='right' caption='[[6bct]], [[Resolution|resolution]] 2.73Å' scene=''> | + | <StructureSection load='6bct' size='340' side='right'caption='[[6bct]], [[Resolution|resolution]] 2.73Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[6bct]] is a 3 chain structure with sequence from [http://en.wikipedia.org/wiki/Atcc_58100 Atcc 58100]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6BCT OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6BCT FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[6bct]] is a 3 chain structure with sequence from [https://en.wikipedia.org/wiki/Leptographium_truncatum Leptographium truncatum]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6BCT OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6BCT FirstGlance]. <br> |
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=CA:CALCIUM+ION'>CA</scene></td></tr> | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.73Å</td></tr> |
| - | <tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">HEG fusion, rps3 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=330483 ATCC 58100])</td></tr> | + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CA:CALCIUM+ION'>CA</scene></td></tr> |
| - | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6bct FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6bct OCA], [http://pdbe.org/6bct PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=6bct RCSB], [http://www.ebi.ac.uk/pdbsum/6bct PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=6bct ProSAT]</span></td></tr> | + | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=6bct FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6bct OCA], [https://pdbe.org/6bct PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6bct RCSB], [https://www.ebi.ac.uk/pdbsum/6bct PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6bct ProSAT]</span></td></tr> |
| | </table> | | </table> |
| - | <div style="background-color:#fffaf0;">
| + | == Function == |
| - | == Publication Abstract from PubMed == | + | [https://www.uniprot.org/uniprot/C7SWF3_9PEZI C7SWF3_9PEZI] |
| - | LAGLIDADG homing endonucleases (meganucleases) are site-specific mobile endonucleases that can be adapted for genome-editing applications. However, one problem when reprogramming meganucleases on non-native substrates is indirect readout of DNA shape and flexibility at the central 4 bases where cleavage occurs. To understand how the meganuclease active site regulates DNA cleavage, we used functional selections and deep sequencing to profile the fitness landscape of 1600 I-LtrI and I-OnuI active site variants individually challenged with 67 substrates with central 4 base substitutions. The wild-type active site was not optimal for cleavage on many substrates, including the native I-LtrI and I-OnuI targets. Novel combinations of active site residues not observed in known meganucleases supported activity on substrates poorly cleaved by the wild-type enzymes. Strikingly, combinations of E or D substitutions in the two metal-binding residues greatly influenced cleavage activity, and E184D variants had a broadened cleavage profile. Analyses of I-LtrI E184D and the wild-type proteins co-crystallized with the non-cognate AACC central 4 sequence revealed structural differences that correlated with kinetic constants for cleavage of individual DNA strands. Optimizing meganuclease active sites to enhance cleavage of non-native central 4 target sites is a straightforward addition to engineering workflows that will expand genome-editing applications.
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| - | Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases.,McMurrough TA, Brown CM, Zhang K, Hausner G, Junop MS, Gloor GB, Edgell DR Nucleic Acids Res. 2018 Oct 24. pii: 5144155. doi: 10.1093/nar/gky976. PMID:30357419<ref>PMID:30357419</ref>
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| - | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br>
| + | |
| - | </div>
| + | |
| - | <div class="pdbe-citations 6bct" style="background-color:#fffaf0;"></div>
| + | |
| - | == References ==
| + | |
| - | <references/>
| + | |
| | __TOC__ | | __TOC__ |
| | </StructureSection> | | </StructureSection> |
| - | [[Category: Atcc 58100]] | + | [[Category: Large Structures]] |
| - | [[Category: Brown, C]] | + | [[Category: Leptographium truncatum]] |
| - | [[Category: Edgell, D R]] | + | [[Category: Brown C]] |
| - | [[Category: Gloor, G B]] | + | [[Category: Edgell DR]] |
| - | [[Category: Junop, M]] | + | [[Category: Gloor GB]] |
| - | [[Category: McMurrough, T A]] | + | [[Category: Junop M]] |
| - | [[Category: Zhang, K]] | + | [[Category: McMurrough TA]] |
| - | [[Category: Hydrolase]]
| + | [[Category: Zhang K]] |
| - | [[Category: Hydrolase-dna complex]]
| + | |
| - | [[Category: Nucleic acid]]
| + | |
