2IMW: Mechanism of Template-Independent Nucleotide Incorporation Catalyzed by a Template-Dependent DNA Polymerase

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.
PDB ID: 2IMWDownload
MMDB ID: 82996
PDB Deposition Date: 2006/10/5
Updated in MMDB: 2012/11
Experimental Method:
x-ray diffraction
Resolution: 2.05  Å
Source Organism:
Sulfolobus solfataricus
Similar Structures:
Biological Unit for 2IMW: trimeric; determined by author
Molecular Components in 2IMW
Label Count Molecule
Protein (1 molecule)
DNA Polymerase IV(Gene symbol: SSO_RS11880)
Molecule annotation
Nucleotides(2 molecules)
Molecule annotation
Molecule annotation
Chemicals (9 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB