Modulation of the Structure, Catalytic Activity, and Fidelity of African Swine Fever Virus DNA Polymerase X by a Reversible Disulfide Switch*

AUTOR(ES)
FONTE

American Society for Biochemistry and Molecular Biology

RESUMO

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 β, 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 β, 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 1H-15N correlated NMR, we unambiguously demonstrated the slow interconversion of pol X between a reduced (pol Xred) and an oxidized form (pol Xox), confirmed by mass spectrometry. Steady-state kinetic analysis revealed that pol Xox, with a disulfide bond between Cys-81 and Cys-86, has ∼10-fold lower fidelity than pol Xred during dNTP insertion opposite a template G. The disulfide linkage is located between two β-strands in the palm domain, near the putative dNTP-binding site. Structural alignment of pol X with a pol β 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.

ACESSO AO ARTIGO

Documentos Relacionados