Plasmodium falciparum specific helicase 3 is nucleocytoplasmic protein and unwinds DNA duplex in 3′ to 5′ direction

Abstract

Plasmodium falciparum is responsible for most dangerous and prevalent form of malaria. The emergence of multi drug resistant parasite hindered the prevention of malaria burden worldwide. Helicases are omnipresent enzymes, which play important role in nucleic acid metabolism and can be used as potential targets for development of novel therapeutics. The genome wide analysis of P. falciparum 3D7 strain revealed some novel parasite specific helicases, which are not present in human host. Here we report the detailed biochemical characterization of P. falciparum parasite specific helicase 3 (PfPSH3). The characteristic ATPase and helicase activities of PfPSH3 reside in its N-terminal region (PfPSH3N) as it contains all the conserved signature motifs whereas the C-terminal does not show any detectable biochemical activity. PfPSH3N also shows DNA helicase activity in the 3′–5′ direction. The immunofluorescence microscopy results show that PSH3 is localized in nucleus as well as in cytoplasm during different stages such as trophozoite and early schizont stages of intraerythrocytic development. This report sets the foundation for further study of parasite specific helicases and will be helpful in understanding the parasite biology.

Biochemical characterization of Plasmodium falciparum parasite specific helicase 1 (PfPSH1)

“…The subcellular localization of PfPSH1 was studied with the help of immunofluorescence microscopy, and it is majorly present in the cytoplasm and partially in the nucleus of the parasite in all the intraerythrocytic developmental stages. It has been previously reported that PfPSH2 and PfPSH3 are localized in both nucleus and cytoplasm in intraerythrocytic developmental stages .…”

“…PfPSH1CM shows loss in ATPase activity, and similarly, helicase activity of the mutant was also abrogated. PfPSH1 is unique in requirement of nucleotide triphosphate when compared to other PSHs (PfPSH2 and PfPSH3); it prefers CTP more than UTP and ATP to unwind DNA, whereas PfPSH2 and PfPSH3 prefer only ATP [19,20]. Despite having similar core and signature motifs various helicases behave differently, previous reports suggest that PfWrn prefers GTP than ATP to unwind DNA and PfUvrD has no preference, and it can unwind well with all four NTPs [32,35].…”

“…The smears of all three developmental stages of parasite were fixed using the prechilled methanol for 20 min at À80°C. The blocking was done using 4% BSA in PBS (19) in a humid chamber at 37°C for 2 h. The slides were washed with PBS and incubated with anti-PfPSH1N antibodies (raised in mice) at 1 : 50 dilution in PBS containing 3% BSA for 2 h at 37°C. The slides were washed two times with PBST (PBS, 0.5% Tween 20) and once with PBS for 5 min each to remove the unbound antibodies.…”

“…PfPSH2 has bipolar direction specificity, but PfPSH3 exhibits only 3 0 -5 0 direction-specific activity. The localization studies suggest that PfPSH2 is mainly localized in the cytoplasm, but the localization of PfPSH3 is nucleocytoplasmic [19,20].…”

“…Our previous studies have shown that there are three parasite‐specific helicases (PSH) present in P. falciparum . In recent studies, we have reported the detailed characterization of two PSH (PfPSH2 and PfPSH3) . The biochemical characterization shows that both PfPSH2 and PfPSH3 are DNA‐dependent ATPases, whereas PfPSH2 also shows RNA‐dependent ATPase activity.…”