5W6Q

Structural basis for recognition of artificial DNA by an evolved KlenTaq variant

Summary for 5W6Q

• Entry DOI: 10.2210/pdb5w6q/pdb
• Descriptor: DNA polymerase I, thermostable, DNA (5'-D(*GP*AP*CP*CP*AP*CP*GP*GP*CP*GP*CP*(1W5))-3'), DNA (5'-D(P*GP*(1WA)P*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3'), ... (4 entities in total)
• Functional Keywords: protein-dna, aegis, unnatural base pair, host-guest system, transferase-dna complex, dna binding protein, dna binding protein-dna complex, dna binding protein/dna
• Biological source: Thermus aquaticus; More
• Total number of polymer chains: 9
• Total formula weight: 205700.68

Authors

Singh, I.,Georgiadis, M.M. (deposition date: 2017-06-16, release date: 2018-07-18, Last modification date: 2023-10-04)

Primary citation

Singh, I.,Laos, R.,Hoshika, S.,Benner, S.A.,Georgiadis, M.M.
Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide.
Nucleic Acids Res., 46:7977-7988, 2018

PubMed 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.

PubMed: 29986111
DOI: 10.1093/nar/gky552

Experimental method

X-RAY DIFFRACTION (2.66 Å)