RESUMEN
Plasmodium falciparum topoisomerase VIB (TopoVIB)-TopoVIA (TopoVIB-VIA) complex can be potentially exploited as a drug target against malaria due to its absence from the human genome. Previous work in our laboratory has suggested that P. falciparum TopoVIB (PfTopoVIB) might be a target of radicicol since treatment of parasite cultures with this antibiotic is associated with upregulation of Plasmodium TopoVIB at the transcript level as well as at the protein level. Further studies demonstrated that radicicol treatment impaired mitochondrial replication of human malaria parasite P. falciparum. However, the technical challenge associated with the expression of the above protein complex hampered its functional characterization. Using Saccharomyces cerevisiae as a heterologous system, we expressed PfTopoVIB (Myc-tagged) and PfTopoVIA (Flag-tagged) (PfTopoVIB-VIA) proteins. Yeast two-hybrid analysis showed the formation of PfTopoVIB homodimers and PfTopoVIB/PfTopoVIA heteromers. Our study demonstrated that PfTopoVIB and PfTopoVIA together can rescue the lethal phenotype of yeast ΔtopoII mutants, whereas Plasmodium topoisomerase VIB alone cannot. Using yeast cell-free extracts harboring the PfTopoVIB-VIA protein complex, we have performed a decatenation assay and observed that PfTopoVIB-VIA can decatenate DNA in an ATP- and Mg(2+)-dependent manner. The specificity of this enzyme is established by abrogation of its activity in the presence of PfTopoVIB-specific antibody. Our study results show that radicicol and etoposide can specifically inhibit PfTopoVIB-VIA decatenation activity whereas the gyrase inhibitor novobiocin cannot. Such a yeast-based assay system can be employed in screening specific inhibitors against Plasmodium VIB-VIA. IMPORTANCE In this study we characterize topoisomerase VI from Plasmodium falciparum using genetic and biochemical approaches. We use various inhibitors and identify radicicol as a specific inhibitor of its decatenation activity. We establish a very simple and economical biochemical assay system that can be exploited to screen inhibitors of PfTopoVI.
RESUMEN
Malaria parasites survive through repairing a plethora of DNA double-stranded breaks (DSBs) experienced during their asexual growth. In Plasmodiumâ Rad51 mediated homologous recombination (HR) mechanism and homology-independent alternative end-joining mechanism have been identified. Here we address whether loss of HR activity can be compensated by other DSB repair mechanisms. Creating a transgenic Plasmodium line defective in HR function, we demonstrate that HR is the most important DSB repair pathway in malarial parasite. Using mouse malaria model we have characterized the dominant negative effect of PfRad51(K143R) mutant on Plasmodiumâ DSB repair and host-parasite interaction. Our work illustrates that Plasmodium berghei harbouring the mutant protein (PfRad51(K143R)) failed to repair DSBs as evidenced by hypersensitivity to DNA-damaging agent. Mice infected with mutant parasites lived significantly longer with markedly reduced parasite burden. To better understand the effect of mutant PfRad51(K143R) on HR, we used yeast as a surrogate model and established that the presence of PfRad51(K143R) completely inhibited DNA repair, gene conversion and gene targeting. Biochemical experiment confirmed that very low level of mutant protein was sufficient for complete disruption of wild-type PfRad51 activity. Hence our work provides evidence that HR pathway of Plasmodium could be efficiently targeted to curb malaria.