Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide

Isha Singh, Roberto Laos, Shuichi Hoshika, Steven A. Benner, Millie Georgiadis

Research output: Contribution to journalArticle

Abstract

The next challenge in synthetic biology is to be able to replicate synthetic nucleic acid sequences efficiently. The synthetic pair, 2-amino-8-(1-beta-D-2- deoxyribofuranosyl) imidazo [1,2-a]-1,3,5-triazin-[8H]-4-one (trivially designated P) with 6-amino-3-(2-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one (trivially designated Z), is replicated by certain Family A polymerases, albeit with lower efficiency. Through directed evolution, we identified a variant KlenTaq polymerase (M444V, P527A, D551E, E832V) that incorporates dZTP opposite P more efficiently than the wild-type enzyme. Here, we report two crystal structures of this variant KlenTaq, a post-incorporation complex that includes a template-primer with P:Z trapped in the active site (binary complex) and a pre-incorporation complex with dZTP paired to template P in the active site (ternary complex). In forming the ternary complex, the fingers domain exhibits a larger closure angle than in natural complexes but engages the template-primer and incoming dNTP through similar interactions. In the binary complex, although many of the interactions found in the natural complexes are retained, there is increased relative motion of the thumb domain. Collectively, our analyses suggest that it is the post-incorporation complex for unnatural substrates that presents a challenge to the natural enzyme and that more efficient replication of P:Z pairs requires a more flexible polymerase.

Original languageEnglish (US)
Pages (from-to)7977-7988
Number of pages12
JournalNucleic Acids Research
Volume46
Issue number15
DOIs
StatePublished - Jan 1 2018
Externally publishedYes

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DNA-Directed DNA Polymerase
Catalytic Domain
Nucleotides
Synthetic Biology
Thumb
Enzymes
Nucleic Acids
Fingers
imidazo(1,2-a)-1,3,5-triazin-4(8H)one
5-amino-1-(2'-deoxy-beta-ribofuranosyl)imidazole-4-carboxamide-5'-triphosphate
2-amino-3-nitropyridin-6-one

ASJC Scopus subject areas

  • Genetics

Cite this

Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide. / Singh, Isha; Laos, Roberto; Hoshika, Shuichi; Benner, Steven A.; Georgiadis, Millie.

In: Nucleic Acids Research, Vol. 46, No. 15, 01.01.2018, p. 7977-7988.

Research output: Contribution to journalArticle

Singh, Isha ; Laos, Roberto ; Hoshika, Shuichi ; Benner, Steven A. ; Georgiadis, Millie. / Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide. In: Nucleic Acids Research. 2018 ; Vol. 46, No. 15. pp. 7977-7988.
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