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

Lamarche, Brandon James

Abstract Details

2005, Doctor of Philosophy, Ohio State University, Chemistry.

The African Swine Fever Virus (ASFV) is a cytoplasmic DNA virus that causes a potentially lethal disease in domestic pigs. Antigenic differences among field isolates indicate that ASFV exists as a diverse population of serotypes in some regions of Africa, and restriction fragment length polymorphisms suggest that this diversity may arise from point mutations.

Consistent with its intracellular location, ASFV encodes three DNA repair proteins: a 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. However, 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 generated. The work described herein was undertaken to assess whether such a system exists.

We demonstrate 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.

Having established a complete viral abasic site repair system, we subsequently assess its capacity for mutagenesis. The catalytic efficiency of nick sealing by ASFV DNA ligase is 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 are incubated with both ASFV DNA ligase and ASFV AP endonuclease, the ligation activity competes very effectively with the 3’→5’ exonuclease editing activity – supporting the hypothesis that error-prone repair of the ASFV genome may facilitate its diversification.

Ming-Daw Tsai (Advisor)
Dehua Pei (Other)
Ross Dalbey (Other)
139 p.

Recommended Citations

Citations

  • Lamarche, B. J. (2005). Error-prone DNA repair in the African swine fever virus: characterization of six abasic site processing activities and evidence for a mutagenic function [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1117656158

    APA Style (7th edition)

  • Lamarche, Brandon. Error-prone DNA repair in the African swine fever virus: characterization of six abasic site processing activities and evidence for a mutagenic function. 2005. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1117656158.

    MLA Style (8th edition)

  • Lamarche, Brandon. "Error-prone DNA repair in the African swine fever virus: characterization of six abasic site processing activities and evidence for a mutagenic function." Doctoral dissertation, Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1117656158

    Chicago Manual of Style (17th edition)