Cloning and characterization of DNA polymerase eta from Trypanosoma cruzi: roles for translesion bypass of oxidative damage.
We report the cloning and characterization of the DNA polymerase eta gene from Trypanosoma cruzi (TcPoleta), the causative agent of Chagas disease. This protein, which can bypass cyclobutane pyrimidine dimers, contains motifs that are conserved between Y family polymerases. In vitro assays showed that the recombinant protein is capable of synthesizing DNA in undamaged primer-templates. Intriguingly, T. cruzi overexpressing TcPoleta does not increase its resistance to UV-light (with or without caffeine) or cisplatin, despite the ability of the protein to enhance UV resistance in a RAD30 mutant of Saccharomyces cerevisiae. Parasites overexpressing TcPoleta are also unable to restore growth after treatment with zeocin or gamma irradiation. T. cruzi overexpressing TcPoleta are more resistant to treatment with hydrogen peroxide (H(2)O(2)) compared to nontransfected cells. The observed H(2)O(2) resistance could be associated with its ability to bypass 8-oxoguanine lesions in vitro. The results presented here suggest that TcPoleta is able to bypass UV and oxidative lesions. However the overexpression of the gene only interferes in response to oxidative lesions, possibly due to the presence of these lesions during the S phase.
de Moura
Michelle Barbi
MB
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, MG, Brazil.
Schamber-Reis
Bruno Luiz Fonseca
BL
Passos Silva
Danielle Gomes
DG
Rajão
Matheus Andrade
MA
Macedo
Andréa Mara
AM
Franco
Glória Regina
GR
Pena
Sérgio Danilo Junho
SD
Teixeira
Santuza Maria Ribeiro
SM
Machado
Carlos Renato
CR
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism / Q000528:radiation effects; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D015800:Protozoan Proteins; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases