2imw
From Proteopedia
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[[Image:2imw.gif|left|200px]] | [[Image:2imw.gif|left|200px]] | ||
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'''Mechanism of Template-Independent Nucleotide Incorporation Catalyzed by a Template-Dependent DNA Polymerase''' | '''Mechanism of Template-Independent Nucleotide Incorporation Catalyzed by a Template-Dependent DNA Polymerase''' | ||
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[[Category: Ling, H.]] | [[Category: Ling, H.]] | ||
[[Category: Yang, W.]] | [[Category: Yang, W.]] | ||
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| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 07:40:20 2008'' | |
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Revision as of 04:40, 4 May 2008
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. 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 Page seeded by OCA on Sun May 4 07:40:20 2008
