2imw
From Proteopedia
(New page: 200px<br /><applet load="2imw" size="450" color="white" frame="true" align="right" spinBox="true" caption="2imw, resolution 2.050Å" /> '''Mechanism of Templa...) |
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| - | [[Image:2imw.gif|left|200px]]<br /><applet load="2imw" size=" | + | [[Image:2imw.gif|left|200px]]<br /><applet load="2imw" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="2imw, resolution 2.050Å" /> | caption="2imw, resolution 2.050Å" /> | ||
'''Mechanism of Template-Independent Nucleotide Incorporation Catalyzed by a Template-Dependent DNA Polymerase'''<br /> | '''Mechanism of Template-Independent Nucleotide Incorporation Catalyzed by a Template-Dependent DNA Polymerase'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Numerous template-dependent DNA polymerases are capable of catalyzing | + | Numerous template-dependent DNA polymerases are capable of catalyzing template-independent nucleotide additions onto blunt-end DNA. Such non-canonical activity has been hypothesized to increase the genomic hypermutability of retroviruses including human immunodeficiency viruses. Here, we employed pre-steady state kinetics and X-ray crystallography to establish a mechanism for blunt-end additions catalyzed by Sulfolobus solfataricus Dpo4. Our kinetic studies indicated that the first blunt-end dATP incorporation was 80-fold more efficient than the second, and among natural deoxynucleotides, dATP was the preferred substrate due to its stronger intrahelical base-stacking ability. Such base-stacking contributions are supported by the 41-fold higher ground-state binding affinity of a nucleotide analog, pyrene nucleoside 5'-triphosphate, which lacks hydrogen bonding ability but possesses four conjugated aromatic rings. A 2.05 A resolution structure of Dpo4*(blunt-end DNA)*ddATP revealed that the base and sugar of the incoming ddATP, respectively, stack against the 5'-base of the opposite strand and the 3'-base of the elongating strand. This unprecedented base-stacking pattern can be applied to subsequent blunt-end additions only if all incorporated dAMPs are extrahelical, leading to predominantly single non-templated dATP incorporation. |
==About this Structure== | ==About this Structure== | ||
| - | 2IMW is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Sulfolobus_solfataricus Sulfolobus solfataricus] with CA, DAD and EDO as [http://en.wikipedia.org/wiki/ligands ligands]. 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:// | + | 2IMW is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Sulfolobus_solfataricus Sulfolobus solfataricus] with <scene name='pdbligand=CA:'>CA</scene>, <scene name='pdbligand=DAD:'>DAD</scene> and <scene name='pdbligand=EDO:'>EDO</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. 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=2IMW OCA]. |
==Reference== | ==Reference== | ||
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[[Category: blunt end dna y-family polymerase dna replication]] | [[Category: blunt end dna y-family polymerase dna replication]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:54:09 2008'' |
Revision as of 15:54, 21 February 2008
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Mechanism of Template-Independent Nucleotide Incorporation Catalyzed by a Template-Dependent DNA Polymerase
Overview
Numerous template-dependent DNA polymerases are capable of catalyzing template-independent nucleotide additions onto blunt-end DNA. Such non-canonical activity has been hypothesized to increase the genomic hypermutability of retroviruses including human immunodeficiency viruses. Here, we employed pre-steady state kinetics and X-ray crystallography to establish a mechanism for blunt-end additions catalyzed by Sulfolobus solfataricus Dpo4. Our kinetic studies indicated that the first blunt-end dATP incorporation was 80-fold more efficient than the second, and among natural deoxynucleotides, dATP was the preferred substrate due to its stronger intrahelical base-stacking ability. Such base-stacking contributions are supported by the 41-fold higher ground-state binding affinity of a nucleotide analog, pyrene nucleoside 5'-triphosphate, which lacks hydrogen bonding ability but possesses four conjugated aromatic rings. A 2.05 A resolution structure of Dpo4*(blunt-end DNA)*ddATP revealed that the base and sugar of the incoming ddATP, respectively, stack against the 5'-base of the opposite strand and the 3'-base of the elongating strand. This unprecedented base-stacking pattern can be applied to subsequent blunt-end additions only if all incorporated dAMPs are extrahelical, leading to predominantly single non-templated dATP incorporation.
About this Structure
2IMW is a Single protein structure of sequence from Sulfolobus solfataricus with , and as ligands. Active as DNA-directed DNA polymerase, with EC number 2.7.7.7 Full crystallographic information is available from OCA.
Reference
Mechanism of template-independent nucleotide incorporation catalyzed by a template-dependent DNA polymerase., Fiala KA, Brown JA, Ling H, Kshetry AK, Zhang J, Taylor JS, Yang W, Suo Z, J Mol Biol. 2007 Jan 19;365(3):590-602. Epub 2006 Oct 7. PMID:17095011
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