Structural basis for C2'-methoxy recognition by DNA polymerases and function improvement.
J Mol Biol
; : 168744, 2024 Aug 13.
Article
em En
| MEDLINE
| ID: mdl-39147125
ABSTRACT
DNA modified with C2'-methoxy (C2'-OMe) greatly enhances its resistance to nucleases, which is beneficial for the half-life of aptamers and DNA nanomaterials. Although the unnatural DNA polymerases capable of incorporating C2'-OMe modified nucleoside monophosphates (C2'-OMe-NMPs) were engineered via directed evolution, the detailed molecular mechanism by which an evolved DNA polymerase recognizes C2'-OMe-NTPs remains poorly understood. Here, we present the crystal structures of the evolved Stoffel fragment of Taq DNA polymerase SFM4-3 processing the C2'-OMe-GTP in different states. Our results reveal the structural basis for recognition of C2'-methoxy by SFM4-3. Based on the analysis of other mutated residues in SFM4-3, a new Stoffel fragment variant with faster catalytic rate and stronger inhibitor-resistance was obtained. In addition, the capture of a novel pre-insertion co-existing with template 5'-overhang stacking conformation provides insight into the catalytic mechanism of Taq DNA polymerase.
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MEDLINE
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En
Ano de publicação:
2024
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Article