Characterization of a functional DNA repair enzyme from Plasmodium falciparum that displays glycosylase and AP-lyase activity

Throughout its residence within the red blood cell the malaria parasite must cope with a significant amount of oxidative stress that results in the increased production of reactive oxygen species. In order to prevent these reactive oxygen species from causing deleterious mutations to its genome the parasite must employ a number of effective strategies for repairing its DNA. We have characterized an Nth/Endonuclease III homologue protein in P. falciparum (PfNth) that effectively recognizes and excises oxidatively damaged DNA as part of the base excision repair pathway. A truncated PfNth [PfNth(s)] protein was produced in E. coli and shown to have both DNA glycosylase and AP-lyase activity specifically directed towards excision of oxidatively damaged DNA. Analysis of the PfNth genomic sequence from seven geographically distinct P. falciparum isolates revealed variations involving a ten amino acid sequence repeat present in the central portion of the protein, as well as a number of nonsynonymous single nucleotide polymorphisms. We tested the possibility that these sequence variations may affect the ability of individual strains to recognize and excise oxidatively damaged DNA thus contributing to the frequency with which drug resistance may arise. However, the biochemical activity of the truncated PfNth(s) from the isolates tested was not affected by the variations in the genomic sequence.; In addition, the effect of exogenous oxidative stress on the transcriptome of the blood stage parasite was examined. The exogenous stress administered to the parasites came in the form of a known pro-oxidant antimalarial drug, artemisinin and an antimalarial drug, atovaquone, which we believe may act as a pro-oxidant. The compounds buthionine sulfoximine, which depletes intracellular levels of glutathione and glucose oxidase, which increases production of hydrogen peroxide, were also tested. We were specifically looking at differences in the transcript levels for genes whose products are involved in DNA repair and antioxidant redox systems. However, over a 4-hour period of treatment with the aforementioned compounds there were no significant changes in the transcript levels for any of these genes. In fact, there were no significant changes in the level of transcription for most genes during this 4-hour period. This is a strikingly different observation than what has been observed for other organisms, where gene expression changes occur within minutes in response to changes in growth conditions such as increased oxidative stress.