Authors

D García-Caballero¹; G Pérez-Moreno¹; L M Ruiz-Pérez¹; A E Vidal¹; D González-Pacanowska¹;  ¹ Instituto de Parasitologia y Biomedicina "Lopez-Neyra", CSIC. Granada, Spain

Discussion

Hypoxanthine (Hx) may arise in DNA as a result of oxidative deamination of adenine or misincorporation of dITP during replication. On the other hand, the occurrence of Hx in RNA is considered a normal and essential modification induced by specific adenosine deaminases acting on RNA. Hx is mainly removed from DNA by specific DNA glycosylases through the Base Excision Repair pathway. In prokaryotes, Endonuclease V (EndoV) can recognize and cleave Hx in DNA, and may be initiating Hx removal by an Alternative Excision Repair pathway. In contrast, human EndoV preferentially cleaves Hx-containing RNA substrates, suggesting a role in RNA metabolism. We have purified and characterized the catalytic properties of the EndoV encoded by Trypanosoma brucei (TbEndoV). In vitro, TbEndoV efficiently processes Hx-containing single-stranded RNA oligonucleotides, including A to Hx-edited tRNA-like substrates. However, a weak activity was observed over Hx-containing DNA, except when a ribonucleotide was placed 3’ to the lesion. Analysis by immunofluorescence microscopy indicates that TbEndoV localizes mainly in the cytosol. The enzyme appears to be not essential in bloodstream forms where the two EndoV copies could be knocked out. In contrast, double allele inactivation could not be achieved in procyclic forms while protein depletion by RNA interference led to impaired growth and defects in cell cycle progression, suggesting a specific role for TbEndoV in this life stage.