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

• Published in: Journal of molecular biology Vol. 365; no. 3; pp. 590 - 602
• Main Authors: Fiala, Kevin A., Brown, Jessica A., Ling, Hong, Kshetry, Ajay K., Zhang, Jun, Taylor, John-Stephen, Yang, Wei, Suo, Zucai
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 19.01.2007
• Subjects: Binding Sites; blunt-end addition; Catalysis; Crystallography, X-Ray; Deoxyadenine Nucleotides - metabolism; DNA - biosynthesis; DNA Polymerase beta - chemistry; DNA Polymerase beta - metabolism; Dpo4; Electrophoresis, Polyacrylamide Gel; Electrophoretic Mobility Shift Assay; Kinetics; Models, Molecular; Nucleotides - chemistry; Nucleotides - metabolism; pre-steady state kinetics; Protein Structure, Secondary; pyrene nucleoside 5′-triphosphate; Pyrenes - chemistry; Pyrenes - metabolism; Retrovirus; Substrate Specificity; Sulfolobus solfataricus; Sulfolobus solfataricus - enzymology; Templates, Genetic; X-ray crystal structure; BPDE; HIV-1; pyrene nucleoside 5′-triphosphate; Dpo4; blunt-end addition; dPTP; pre-steady state kinetics; dNTP; X-ray crystal structure
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2006.10.008

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.