| Summary: | Three constructs of African Swine Fever Virus DNA polymerase X (pol X) were expressed and purified. Buffers containing sodium acetate, sodium cacodylate or 0.5 M NaCl provided good solubility for the enzyme at micromolar concentration, which was required for biophysical studies.
The transformation between the oxidized and reduced forms of the enzyme was monitored by nuclear magnetic resonance (NMR). Biochemical assays revealed that the oxidized form of the enzyme showed activity with template-primer substrate, whereas the reduced form was only weakly active.
NMR chemical shift assignments for the pol X - deoxyguanosine triphosphate complex were completed. Based on these assignments, chemical shift perturbations of several dNTP binary complexes were mapped to the pol X structure, revealing interactions with the â strands 4, 6, 8-10 and á-helices B and D. Isothermal titration calorimetry (ITC) and NMR measurements suggested a different interaction mode for purines and pyrimidines with pol X which was supported by the difference in dissociation constants (Kd) for purines, 2-10 µM and pyrimidines, 40-50 µM.
Dissociation constants in the high nanomolar to low micromolar range were determined for double hairpin and template-primer oligodeoxynucleotide sequences. Binding interactions of oligodeoxynucleotides were mapped to the structure of pol X by analysis of amide chemical shift perturbations, displaying perturbations at the C-terminal end of á-helix E, â-sheet 11 and 12, and at the sub-domain binding interface. Based on these observations, a model analogous to pol â was proposed, where a bent DNA would span the enzyme between the C-terminal side of áE and áC, or áE and the subdomain interface, possibly moving between the two positions, while the enzyme undergoes further conformational changes.
|
|---|
