DNA polymerase kappa from Trypanosoma cruzi localizes to the mitochondria, bypasses 8-oxoguanine lesions and performs DNA synthesis in a recombination intermediate.
DNA polymerase kappa (Pol kappa) is a low-fidelity polymerase that has the ability to bypass several types of lesions. The biological role of this enzyme, a member of the DinB subfamily of Y-family DNA polymerases, has remained elusive. In this report, we studied one of the two copies of Pol kappa from the protozoan Trypanosoma cruzi (TcPol kappa). The role of this TcPol kappa copy was investigated by analysing its subcellular localization, its activities in vitro, and performing experiments with parasites that overexpress this polymerase. The TcPOLK sequence has the N-terminal extension which is present only in eukaryotic DinB members, but its C-terminal region is more similar to prokaryotic and archaeal counterparts since it lacks C(2)HC motifs and PCNA interaction domain. Our results indicate that in contrast to its previously described orthologues, this polymerase is localized to mitochondria. The overexpression of TcPOLK increases T. cruzi resistance to hydrogen peroxide, and in vitro polymerization assays revealed that TcPol kappa efficiently bypasses 8-oxoguanine lesions. Remarkably, our results also demonstrate that the DinB subfamily of polymerases can participate in homologous recombination, based on our findings that TcPol kappa increases T. cruzi resistance to high doses of gamma irradiation and zeocin and can catalyse DNA synthesis within recombination intermediates.
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Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Caixa Postal 486, Belo Horizonte 30161-970, MG, Brazil.
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D000595:Amino Acid Sequence; D000818:Animals; D004249:DNA Damage; D004261:DNA Replication; D016054:DNA, Protozoan / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008928:Mitochondria / Q000235:genetics*; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
D016054:DNA, Protozoan; D015800:Protozoan Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D006861:Hydrogen Peroxide; D004259:DNA-Directed DNA Polymerase