This work describes the multi-faceted characterization of DNA polymerase X (Pol X) from African Swine Fever Virus (ASFV), the smallest known DNA polymerase belonging to the X family of DNA polymerases. Early kinetic characterization of Pol X showed it to be highly error-prone and it was hypothesized to contribute to the viral hypermutagenicity. However, because some reports discredited the low-fidelity of Pol X, we have performed extensive analyses of Pol X fidelity under a varied array of assay conditions and DNA substrates. Our results show that Pol X is indeed low fidelity under all the conditions examined. In order to elucidate a molecular and biological rationale for the extreme error-proneness of Pol X, we have examined its ability to utilize damaged DNA and dNTP substrates using a diverse selection of DNA lesions. Our results show that Pol X possesses moderate lesion bypass capabilities, characterized by efficient bypass of 8-oxo-guanine and blockage by abasic site and thymine glycol lesions in DNA.
Pol X serves as a model system for structural and mechanistic analyses of fidelity of DNA polymerases because of its small size and low fidelity. We have examined the Pol X-catalyzed correct dNTP incorporation in stopped-flow fluorescence assays and show the presence of a non-rate limiting physical step leading to the formation of the catalytically active conformation of the ternary complex, followed by the rate-limiting chemical transformation. Our structural and kinetics analyses of the dG:dGTP mismatch incorporation show that dG:dGTP mismatch adopts a anti:syn conformation, forming a Hoogsteen base pair, in the Pol X active site. Further, our fidelity analyses of the site-directed mutants show that His115 residue is critical for maintaining the low fidelity of Pol X. Towards a complete understanding of its mechanism; we have performed a comprehensive analysis of the order of substrate binding by Pol X. We show that Pol X productively binds dNTP before binding DNA in correct conformation, contrary to the DNA first binding order established for several DNA polymerases. Our findings suggest that low-fidelity DNA polymerases may not require the strictly ordered mechanism of substrate binding utilized by high-fidelity replicative DNA polymerases.