2imw
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Mechanism of Template-Independent Nucleotide Incorporation Catalyzed by a Template-Dependent DNA Polymerase
OverviewOverview
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 StructureAbout 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.
ReferenceReference
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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