Characterization of a DNA base excision repair pathway in the malaria parasite Plasmodium falciparum: From RIPs to repair
The investigations described in this thesis report two novel findings: (1) the identification of a new class of adenine specific single-stranded DNA glycosylases and (2) the first description of DNA repair activities in Plasmodium falciparum, the protozoan parasite that causes human malaria. Chapter One investigated an alternative activity of the plant ribosome-inactivating protein (RIP) gelonin, which was proposed to be responsible for the cytotoxicity seen in RIP treated P. falciparum. To address the issue of a contaminant, preparations of native and bacterial recombinant gelonin were analyzed for nuclease activity. Because the mitochondrial DNA of P. falciparum appeared to be the target of gelonin's cytotoxic activity and contained single-stranded regions, we investigated the activity of gelonin on single stranded DNA. Both the native and recombinant forms of gelonin were found to cause the degradation of single stranded DNA. This activity was modulated by zinc. It is unlikely that the DNase activity of both the native and recombinant protein were due to a contaminant because nuclease activity gel analysis in conjunction with Western blotting using an anti-gelonin antibody indicated that the polypeptide responsible for DNase activity was gelonin. Chapter Two further characterized the nuclease activity of gelonin and compared it to two other RIPs, pokeweed antiviral protein (PAP) and ricin. A more detailed analysis of the nuclease activity found that the cleavage was specific for adenine. Additionally, it was found that the removal of adenine by a single-strand DNA glycosylase activity was the first step in the cleavage of DNA. After the removal of adenine, the DNA was then cleaved, possibly by an associated lyase activity. This activity was unique, as there is no other report of a DNA glycosylase/AP lyase that acts on single stranded DNA. The investigation reported in Chapter Three characterized the repair of uracil mismatches and apurinic/apyrimidinic (AP) sites in DNA by a total cell lysate from P. falciparum. The investigation showed that the lysate was capable of removing uracil from DNA, generating AP sites. The lysate then processed the AP sites with a class II AP endonuclease that hydrolytically cleaved the AP site generating 3′ OH and 5′ dRp moieties. P. falciparum appeared to repair the nicked strand exclusively by a long-patch BER pathway. It is likely that this pathway utilized a flap endonuclease to process the single-stranded DNA that occurs during repair synthesis. This was similar to the processing seen in reconstituted mammalian systems containing FEN-1. It appears that the cell-free lysate from P. falciparum possesses a complete pathway for the repair of uracil damage and AP sites. The identification of this new aspect of parasite biology might open new vistas for the development of new chemotherapeutic targets for treating an old disease. (Abstract shortened by UMI.)
Haltiwanger, Brett Maher, "Characterization of a DNA base excision repair pathway in the malaria parasite Plasmodium falciparum: From RIPs to repair" (1999). ETD Collection for Thomas Jefferson University. AAI9921830.