Use of damaged DNA and dNTP substrates by the error-prone DNA polymerase X from African swine fever virus.
The structural specificity that translesion DNA polymerases often show for a particular class of lesions suggests that the predominant criterion of selection during their evolution has been the capacity for lesion tolerance and that the error-proneness they display when copying undamaged templates may simply be a byproduct of this adaptation. Regardless of selection criteria/evolutionary history, at present both of these properties coexist in these enzymes, and both properties confer a fitness advantage. The repair polymerase, Pol X, encoded by the African swine fever virus (ASFV) is one of the most error-prone polymerases known, leading us to previously hypothesize that it may work in tandem with the exceptionally error-tolerant ASFV DNA ligase to effect viral mutagenesis. Here, for the first time, we test whether the error-proneness of Pol X is coupled with a capacity for lesion tolerance by examining its ability to utilize the types of damaged DNA and dNTP substrates that are expected to be relevant to ASFV. We (i) test Pol X's ability to both incorporate opposite to and extend from ubiquitous oxidative purine (7,8-dihydro-8-oxoguanine), oxidative pyrimidine (5,6-dihydroxy-5,6-dihydrothymine), and noncoding (AP site) lesions, in addition to 5,6-dihydrothymine, (ii) determine the catalytic efficiency and dNTP specificity of Pol X when catalyzing incorporation opposite to, and when extending from, 7,8-dihydro-8-oxoguanine in a template/primer context, and (iii) quantitate Pol X-catalyzed incorporation of the damaged nucleotide 8-oxo-dGTP opposite to undamaged templates in the context of both template/primer and a single-nucleotide gap. Our findings are discussed in light of ASFV biology and the mutagenic DNA repair hypothesis described above.
Kumar
Sandeep
S
Department of Chemistry, The Biophysics Program, The Ohio State University, Columbus, Ohio 43210, USA.
Lamarche
Brandon J
BJ
Tsai
Ming-Daw
MD
D000358:African Swine Fever Virus / Q000737:chemistry / Q000201:enzymology*; D002384:Catalysis; D004249:DNA Damage; D011088:DNA Ligases / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D019143:Evolution, Molecular; D016296:Mutagenesis
D003854:Deoxyribonucleotides; C109534:DNA polymerase X; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases