Modulation of the structure, catalytic activity, and fidelity of African swine fever virus DNA polymerase X by a reversible disulfide switch.

Voehler MW, Eoff RL, McDonald WH, Guengerich FP, Stone MP
J Biol Chem. 2009 284 (27): 18434-44

PMID: 19419958 · PMCID: PMC2709365 · DOI:10.1074/jbc.M109.012542

African swine fever virus polymerase X (pol X) is the smallest DNA polymerase known (174 amino acids), and its tertiary structure resembles the C-terminal half of prototypical X-family pol beta, which includes a catalytic dNTP-binding site (palm domain) and a finger domain. This structural similarity and the presence of viral genes coding for other base excision repair proteins suggest that pol X functions in a manner similar to pol beta, but inconsistencies concerning pol X catalysis have been reported. We examined the structural and functional properties of two forms of pol X using spectroscopic and kinetic analysis. Using (1)H-(15)N correlated NMR, we unambiguously demonstrated the slow interconversion of pol X between a reduced (pol X(red)) and an oxidized form (pol X(ox)), confirmed by mass spectrometry. Steady-state kinetic analysis revealed that pol X(ox), with a disulfide bond between Cys-81 and Cys-86, has approximately 10-fold lower fidelity than pol X(red) during dNTP insertion opposite a template G. The disulfide linkage is located between two beta-strands in the palm domain, near the putative dNTP-binding site. Structural alignment of pol X with a pol beta ternary structure suggests that the disulfide switch may modulate fidelity by altering the ability of the palm domain to align and stabilize the primer terminus and catalytic metal ion for deprotonation of the 3'-OH group and subsequent phosphoryl transfer. Thus, DNA polymerase fidelity is altered by the redox state of the enzyme and its related conformational changes.

MeSH Terms (13)

African Swine Fever Virus Catalysis Disulfides DNA, Viral DNA-Directed DNA Polymerase Enzyme Activation Guanosine Triphosphate Kinetics Nuclear Magnetic Resonance, Biomolecular Oxidation-Reduction Structure-Activity Relationship Substrate Specificity Virus Replication

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