Error-prone DNA repair in the African swine fever virus: Characterization of six abasic site processing activities and evidence for a mutagenic function

The African Swine Fever Virus (ASFV) is a complex (∼150 genes), cytoplasmic, double-stranded DNA virus that causes a potentially lethal disease in domestic pigs. Antigenic differences among field isolates suggest that ASFV exists as a diverse population of serotypes in some regions of Africa. Moreover, restriction fragment length polymorphisms---in the absence of major genome rearrangements---suggest that the genetic diversity of ASFV may arise from point mutations or small insertions/deletions.; Consistent with its intracellular location, ASFV encodes three DNA repair proteins: a repair polymerase, an AP endonuclease, and a DNA ligase. The ASFV repair polymerase, Pol X, is extremely error prone during single nucleotide gap filling, leading others to hypothesize that it might contribute to the genetic variability of ASFV. In order for the error-proneness of Pol X to be biologically relevant it would need to function within a repair system in which each of the components tolerated and/or utilized the mismatched intermediates and products being formed. The work described here was undertaken in order to assess whether such a system exists.; Herein we demonstrate for the first time that ASFV gene E296R is an AP endonuclease, a 3'-phosphodiesterase, and a 3'→5 exonuclease. Pol X and ASFV DNA ligase are both shown to contribute lyase activity towards 5'-2-deoxyribose-5-phosphate. With this complement of activities we demonstrate that ASFV is capable of effecting repair of abasic sites without the need to recruit host factors.; Having established a complete abasic site repair system, we assessed its capacity for mutagenesis. The catalytic efficiency of nick sealing by ASFV DNA ligase was determined for substrates containing all 16 possible base pair combinations at the 3' side of a nick. Our results indicate this enzyme to be the lowest fidelity DNA ligase ever reported---capable of ligating a 3' mismatched nick more efficiently than nicks containing Watson-Crick base pairs. Comparison of the mismatch specificity of Pol X with that of ASFV DNA ligase suggests that the latter may have evolved towards low fidelity for the purpose of generating the broadest possible spectrum of sealed mismatches. In competition experiments where mismatched nicks were incubated with both ASFV DNA ligase and ASFV AP endonuclease, the ligation activity competed very effectively with the 3'→5 ' exonuclease editing activity---supporting the hypothesis that error-prone repair of the ASFV genome may facilitate its diversification.