DNA N-glycosylases Ogg1 and EndoIII as components of base excision repair in Plasmodium falciparum organelles.

The integrity of genomes of the two crucial organelles of the malaria parasite - an apicoplast and mitochondrion in each cell - must be maintained by DNA repair mediated by proteins targeted to these compartments. We explored the localisation and function of Plasmodium falciparum base excision repair (BER) DNA N-glycosylase homologs PfEndoIII and PfOgg1. These N-glycosylases would putatively recognise DNA lesions prior to the action of apurinic/apyrimidinic (AP)-endonucleases. Both Ape1 and Apn1 endonucleases have earlier been shown to function solely in the parasite mitochondrion. Immunofluorescence localisation showed that PfEndoIII was exclusively mitochondrial. PfOgg1 was not seen clearly in mitochondria when expressed as a PfOgg1leader-GFP fusion, although chromatin immunoprecipitation assays showed that it could interact with both mitochondrial and apicoplast DNA. Recombinant PfEndoIII functioned as a DNA N-glycosylase as well as an AP-lyase on thymine glycol (Tg) lesions. We further studied the importance of Ogg1 in the malaria life cycle using reverse genetic approaches in Plasmodium berghei. Targeted disruption of PbOgg1 resulted in loss of 8-oxo-G specific DNA glycosylase/lyase activity. PbOgg1 knockout did not affect blood, mosquito or liver stage development but caused reduced blood stage infection after inoculation of sporozoites in mice. A significant reduction in erythrocyte infectivity by PbOgg1 knockout hepatic merozoites was also observed, thus showing that PbOgg1 ensures smooth transition from liver to blood stage infection. Our results strengthen the view that the Plasmodium mitochondrial genome is an important site for DNA repair by the BER pathway.

A Plasmodium apicoplast-targeted unique exonuclease/FEN exhibits interspecies functional differences attributable to an insertion that alters DNA-binding.

The human malaria parasite Plasmodium falciparum genome is among the most A + T rich, with low complexity regions (LCRs) inserted in coding sequences including those for proteins targeted to its essential relict plastid (apicoplast). Replication of the apicoplast genome (plDNA), mediated by the atypical multifunctional DNA polymerase PfPrex, would require additional enzymatic functions for lagging strand processing. We identified an apicoplast-targeted, [4Fe-4S]-containing, FEN/Exo (PfExo) with a long LCR insertion and detected its interaction with PfPrex. Distinct from other known exonucleases across organisms, PfExo recognized a wide substrate range; it hydrolyzed 5'-flaps, processed dsDNA as a 5'-3' exonuclease, and was a bipolar nuclease on ssDNA and RNA-DNA hybrids. Comparison with the rodent P. berghei ortholog PbExo, which lacked the insertion and [4Fe-4S], revealed interspecies functional differences. The insertion-deleted PfExoΔins behaved like PbExo with a limited substrate repertoire because of compromised DNA binding. Introduction of the PfExo insertion into PbExo led to gain of activities that the latter initially lacked. Knockout of PbExo indicated essentiality of the enzyme for survival. Our results demonstrate the presence of a novel apicoplast exonuclease with a functional LCR that diversifies substrate recognition, and identify it as the candidate flap-endonuclease and RNaseH required for plDNA replication and maintenance.

Mycochemical Profiling and Antioxidant Activity of Two Different Tea Preparations from Lion's Mane Medicinal Mushroom, Hericium erinaceus (Agaricomycetes).

Mushrooms have become the new superfood with their many bioactive metabolites and are potential candidates in the field of herbal medicine. Since not all mushrooms can be consumed whole, their active constituents and therapeutic benefits can be had in the form of beverages specially teas or coffees. In the present study, two forms of teas, infusion and decoction, were prepared from Hericium erinaceus (Bull.) Pers., a very popular mushroom in Chinese medicine. Both forms of tea were studied mycochemically and medicinally and a comparative view was presented on the basis of the findings. The tea preparations were rich in bioactive mycochemicals; interestingly, the infusion contained a higher amount of phenol (1.72 mg gallic acid equivalent/g of dry weight of mushrooms) than decoction (0.28 mg gallic acid equivalent/g of dry weight of mushrooms). Lycopene and ß-carotene were found in very minute amounts. Both infusion and decoction exhibited good free radical scavenging potential, reducing power, and total antioxidant properties. However, the infusion fraction produced overall better results than the decoction fraction. Finally, the results suggest that H. erinaceus is a potent source of natural antioxidant and also can be consumed as a beverage.