Trypanosomes lacking uracil-DNA glycosylase are hypersensitive to antifolates and present a mutator phenotype.
Cells contain low amounts of uracil in DNA which can be the result of dUTP misincorporation during replication or cytosine deamination. Elimination of uracil in the base excision repair pathway yields an abasic site, which is potentially mutagenic unless repaired. The Trypanosoma brucei genome presents a single uracil-DNA glycosylase responsible for removal of uracil from DNA. Here we establish that no excision activity is detected on U:G, U:A pairs or single-strand uracil-containing DNA in uracil-DNA glycosylase null mutant cell extracts, indicating the absence of back-up uracil excision activities. While procyclic forms can survive with moderate amounts of uracil in DNA, an analysis of the mutation rate and spectra in mutant cells revealed a hypermutator phenotype where the predominant events were GC to AT transitions and insertions. Defective elimination of uracil via the base excision repair pathway gives rise to hypersensitivity to antifolates and oxidative stress and an increased number of DNA strand breaks, suggesting the activation of alternative DNA repair pathways. Finally, we show that uracil-DNA glycosylase defective cells exhibit reduced infectivity in vivo demonstrating that efficient uracil elimination is important for survival within the mammalian host.
Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Parque Tecnológico de Ciencias de la Salud, Armilla (Granada), Spain.
Víctor M
VM
Castillo-Acosta
Fernando
F
Aguilar-Pereyra
Antonio E
AE
Vidal
Miguel
M
Navarro
Luis M
LM
Ruiz-Pérez
Dolores
D
González-Pacanowska
D000818:Animals; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D053960:DNA Breaks / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects; D016054:DNA, Protozoan / Q000235:genetics; D005260:Female; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D020862:GC Rich Sequence; D055786:Gene Knockout Techniques; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D009154:Mutation; D010641:Phenotype; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D051981:Uracil-DNA Glycosidase / Q000172:deficiency* / Q000235:genetics
D016054:DNA, Protozoan; D005493:Folic Acid Antagonists; D006861:Hydrogen Peroxide; D051981:Uracil-DNA Glycosidase; D008727:Methotrexate