Untitled

Abstract

DNA polymerase X (Pol X) from the African swine fever virus (ASFV) specifically binds intermediates in the single-nucleotide base-excision repair process, an activity indicative of repair function. In addition, Pol X catalyzes DNA polymerization with low nucleotide-insertion fidelity. The structural mechanisms by which DNA polymerases confer high or low fidelity in DNA polymerization remain to be elucidated. The three-dimensional structure of Pol X has been determined. Unlike other DNA polymerases, Pol X is formed from only a palm and a C-terminal subdomain. Pol X has a novel palm subdomain fold, containing a positively charged helix at the DNA binding surface. Purine deoxynucleoside triphosphate (dNTP) substrates bind between the palm and C-terminal subdomain, at a dNTP-binding helix, and induce a unique conformation in Pol X. The purine dNTP-bound conformation and high binding affinity for dGTP-Mg(2+) of Pol X may contribute to its low fidelity.

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

“…2). The chemical shifts from a freshly prepared pol X sample were in good agreement with the data deposited at the Biological Magnetic Resonance Bank by Maciejewski et al (36,37), but this was no longer the case after 4 -6 months for those shifting peaks. Between measurements, the sample was maintained without further manipulation at 4°C in the standard buffer, containing 5 mM DTT.…”

“…Enzyme purification was as described (36,38), but 20 mM PIPES-Na buffer was used throughout the purification instead of phosphate. The standard NMR buffer contained 20 mM PIPES-Na (pH 6.5), 20 mM MgCl 2 , 0.02% NaN 3 (w/v), 0.5 M NaCl, and 5 mM DTT.…”

“…Both cysteines reside in the palm domain of pol X and are the only cysteines present in the protein. They also present the main difference in the two NMR solution structures that have been published (30,36). One structure was described to contain reduced cysteines (36), and the other structure was reported to contain a disulfide linkage (30).…”

“…They also present the main difference in the two NMR solution structures that have been published (30,36). One structure was described to contain reduced cysteines (36), and the other structure was reported to contain a disulfide linkage (30). The latter structure was based on the sole observation of two downfield-shifted 13 C-␤ cysteine resonances.…”

The Family X DNA Polymerase from Deinococcus radiodurans Adopts a Non-standard Extended Conformation

“…In view of the radically different relative configuration of the PolX Dr domains compared with other family X members, it remains an open question whether the same mechanism is at work here. It is interesting to note that the African swine fever PolX ASFV altogether lacks the finger and 8-kDa domains but is a genuine polymerase (30). Therefore the role of the positioning or even the presence of the individual subdomains on polymerase activities has not definitely been settled.…”