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1x9w
From Proteopedia
(New page: 200px<br /><applet load="1x9w" size="450" color="white" frame="true" align="right" spinBox="true" caption="1x9w, resolution 2.30Å" /> '''T7 DNA polymerase in...) |
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| - | [[Image:1x9w.gif|left|200px]]<br /><applet load="1x9w" size=" | + | [[Image:1x9w.gif|left|200px]]<br /><applet load="1x9w" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1x9w, resolution 2.30Å" /> | caption="1x9w, resolution 2.30Å" /> | ||
'''T7 DNA polymerase in complex with a primer/template DNA containing a disordered N-2 aminofluorene on the template, crystallized with dideoxy-ATP as the incoming nucleotide.'''<br /> | '''T7 DNA polymerase in complex with a primer/template DNA containing a disordered N-2 aminofluorene on the template, crystallized with dideoxy-ATP as the incoming nucleotide.'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The carcinogen 2-acetylaminofluorene forms two major DNA adducts: | + | The carcinogen 2-acetylaminofluorene forms two major DNA adducts: N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and its deacetylated derivative, N-(2'-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF). Although the dG-AAF and dG-AF adducts are distinguished only by the presence or absence of an acetyl group, they have profoundly different effects on DNA replication. dG-AAF poses a strong block to DNA synthesis and primarily induces frameshift mutations in bacteria, resulting in the loss of one or two nucleotides during replication past the lesion. dG-AF is less toxic and more easily bypassed by DNA polymerases, albeit with an increased frequency of misincorporation opposite the lesion, primarily resulting in G --> T transversions. We present three crystal structures of bacteriophage T7 DNA polymerase replication complexes, one with dG-AAF in the templating position and two others with dG-AF in the templating position. Our crystallographic data suggest why a dG-AAF adduct blocks replication more strongly than does a dG-AF adduct and provide a possible explanation for frameshift mutagenesis during replication bypass of a dG-AAF adduct. The dG-AAF nucleoside adopts a syn conformation that facilitates the intercalation of its fluorene ring into a hydrophobic pocket on the surface of the fingers subdomain and locks the fingers in an open, inactive conformation. In contrast, the dG-AF base at the templating position is not well defined by the electron density, consistent with weak binding to the polymerase and a possible interchange of this adduct between the syn and anti conformations. |
==About this Structure== | ==About this Structure== | ||
| - | 1X9W is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Bacteriophage_t7 Bacteriophage t7] and [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with MG as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/DNA-directed_DNA_polymerase DNA-directed DNA polymerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.7 2.7.7.7] Full crystallographic information is available from [http:// | + | 1X9W is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Bacteriophage_t7 Bacteriophage t7] and [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=MG:'>MG</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/DNA-directed_DNA_polymerase DNA-directed DNA polymerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.7 2.7.7.7] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1X9W OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Johnson, D.]] | [[Category: Johnson, D.]] | ||
[[Category: Li, Y.]] | [[Category: Li, Y.]] | ||
| - | [[Category: Richardson, C | + | [[Category: Richardson, C C.]] |
| - | [[Category: Romano, L | + | [[Category: Romano, L J.]] |
[[Category: MG]] | [[Category: MG]] | ||
[[Category: dna polymerase]] | [[Category: dna polymerase]] | ||
| Line 28: | Line 28: | ||
[[Category: replication block]] | [[Category: replication block]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:52:32 2008'' |
Revision as of 13:52, 21 February 2008
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T7 DNA polymerase in complex with a primer/template DNA containing a disordered N-2 aminofluorene on the template, crystallized with dideoxy-ATP as the incoming nucleotide.
Overview
The carcinogen 2-acetylaminofluorene forms two major DNA adducts: N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and its deacetylated derivative, N-(2'-deoxyguanosin-8-yl)-2-aminofluorene (dG-AF). Although the dG-AAF and dG-AF adducts are distinguished only by the presence or absence of an acetyl group, they have profoundly different effects on DNA replication. dG-AAF poses a strong block to DNA synthesis and primarily induces frameshift mutations in bacteria, resulting in the loss of one or two nucleotides during replication past the lesion. dG-AF is less toxic and more easily bypassed by DNA polymerases, albeit with an increased frequency of misincorporation opposite the lesion, primarily resulting in G --> T transversions. We present three crystal structures of bacteriophage T7 DNA polymerase replication complexes, one with dG-AAF in the templating position and two others with dG-AF in the templating position. Our crystallographic data suggest why a dG-AAF adduct blocks replication more strongly than does a dG-AF adduct and provide a possible explanation for frameshift mutagenesis during replication bypass of a dG-AAF adduct. The dG-AAF nucleoside adopts a syn conformation that facilitates the intercalation of its fluorene ring into a hydrophobic pocket on the surface of the fingers subdomain and locks the fingers in an open, inactive conformation. In contrast, the dG-AF base at the templating position is not well defined by the electron density, consistent with weak binding to the polymerase and a possible interchange of this adduct between the syn and anti conformations.
About this Structure
1X9W is a Protein complex structure of sequences from Bacteriophage t7 and Escherichia coli with as ligand. Active as DNA-directed DNA polymerase, with EC number 2.7.7.7 Full crystallographic information is available from OCA.
Reference
Crystal structures of 2-acetylaminofluorene and 2-aminofluorene in complex with T7 DNA polymerase reveal mechanisms of mutagenesis., Dutta S, Li Y, Johnson D, Dzantiev L, Richardson CC, Romano LJ, Ellenberger T, Proc Natl Acad Sci U S A. 2004 Nov 16;101(46):16186-91. Epub 2004 Nov 4. PMID:15528277
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