1g9y: Difference between revisions
New page: left|200px<br /><applet load="1g9y" size="450" color="white" frame="true" align="right" spinBox="true" caption="1g9y, resolution 2.05Å" /> '''HOMING ENDONUCLEASE ... |
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[[Image:1g9y.gif|left|200px]]<br /><applet load="1g9y" size=" | [[Image:1g9y.gif|left|200px]]<br /><applet load="1g9y" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1g9y, resolution 2.05Å" /> | caption="1g9y, resolution 2.05Å" /> | ||
'''HOMING ENDONUCLEASE I-CREI / DNA SUBSTRATE COMPLEX WITH CALCIUM'''<br /> | '''HOMING ENDONUCLEASE I-CREI / DNA SUBSTRATE COMPLEX WITH CALCIUM'''<br /> | ||
==Overview== | ==Overview== | ||
Homing endonucleases, like restriction enzymes, cleave double-stranded DNA | Homing endonucleases, like restriction enzymes, cleave double-stranded DNA at specific target sites. The cleavage mechanism(s) utilized by LAGLIDADG endonucleases have been difficult to elucidate; their active sites are divergent, and only one low resolution cocrystal structure has been determined. Here we report two high resolution structures of the dimeric I-CreI homing endonuclease bound to DNA: a substrate complex with calcium and a product complex with magnesium. The bound metals in both complexes are verified by manganese anomalous difference maps. The active sites are positioned close together to facilitate cleavage across the DNA minor groove; each contains one metal ion bound between a conserved aspartate (Asp 20) and a single scissile phosphate. A third metal ion bridges the two active sites. This divalent cation is bound between aspartate residues from the active site of each subunit and is in simultaneous contact with the scissile phosphates of both DNA strands. A metal-bound water molecule acts as the nucleophile and is part of an extensive network of ordered water molecules that are positioned by enzyme side chains. These structures illustrate a unique variant of a two-metal endonuclease mechanism is employed by the highly divergent LAGLIDADG enzyme family. | ||
==About this Structure== | ==About this Structure== | ||
1G9Y is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Chlamydomonas_reinhardtii Chlamydomonas reinhardtii] with CA as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http:// | 1G9Y is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Chlamydomonas_reinhardtii Chlamydomonas reinhardtii] with <scene name='pdbligand=CA:'>CA</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1G9Y OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Chevalier, B.]] | [[Category: Chevalier, B.]] | ||
[[Category: Monnat, R | [[Category: Monnat, R J.]] | ||
[[Category: Stoddard, B | [[Category: Stoddard, B L.]] | ||
[[Category: CA]] | [[Category: CA]] | ||
[[Category: group i intron]] | [[Category: group i intron]] | ||
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[[Category: nuclease mechanism]] | [[Category: nuclease mechanism]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:47:47 2008'' | ||
Revision as of 13:47, 21 February 2008
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HOMING ENDONUCLEASE I-CREI / DNA SUBSTRATE COMPLEX WITH CALCIUM
OverviewOverview
Homing endonucleases, like restriction enzymes, cleave double-stranded DNA at specific target sites. The cleavage mechanism(s) utilized by LAGLIDADG endonucleases have been difficult to elucidate; their active sites are divergent, and only one low resolution cocrystal structure has been determined. Here we report two high resolution structures of the dimeric I-CreI homing endonuclease bound to DNA: a substrate complex with calcium and a product complex with magnesium. The bound metals in both complexes are verified by manganese anomalous difference maps. The active sites are positioned close together to facilitate cleavage across the DNA minor groove; each contains one metal ion bound between a conserved aspartate (Asp 20) and a single scissile phosphate. A third metal ion bridges the two active sites. This divalent cation is bound between aspartate residues from the active site of each subunit and is in simultaneous contact with the scissile phosphates of both DNA strands. A metal-bound water molecule acts as the nucleophile and is part of an extensive network of ordered water molecules that are positioned by enzyme side chains. These structures illustrate a unique variant of a two-metal endonuclease mechanism is employed by the highly divergent LAGLIDADG enzyme family.
About this StructureAbout this Structure
1G9Y is a Single protein structure of sequence from Chlamydomonas reinhardtii with as ligand. Full crystallographic information is available from OCA.
ReferenceReference
The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites., Chevalier BS, Monnat RJ Jr, Stoddard BL, Nat Struct Biol. 2001 Apr;8(4):312-6. PMID:11276249
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