Mechanism of Template-independent Nucleotide Incorporation Catalyzed by a Template-dependent DNA Polymerase

Kevin A. Fiala, Jessica A. Brown, Hong Ling, Ajay K. Kshetry, Jun Zhang, John Stephen Taylor, Wei Yang, Zucai Suo

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


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 Å 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.

Original languageEnglish
Pages (from-to)590-602
Number of pages13
JournalJournal of Molecular Biology
Issue number3
StatePublished - Jan 19 2007


  • blunt-end addition
  • Dpo4
  • pre-steady state kinetics
  • pyrene nucleoside 5′-triphosphate
  • X-ray crystal structure


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