Plasmodium falciparum UvrD Helicase Translocates in 3′ to 5′ Direction, Colocalizes with MLH and Modulates Its Activity through Physical Interaction

Malaria is a global disease and a major health problem. The control of malaria is a daunting task due to the increasing drug resistance. Therefore, there is an urgent need to identify and characterize novel parasite specific drug targets. In the present study we report the biochemical characterization of parasite specific UvrD helicase from Plasmodium falciparum. The N-terminal fragment (PfUDN) containing UvrD helicase domain, which consists of helicase motifs Q, Ia–Id, II, III and most of motif IV, and the C-terminal fragment (PfUDC1) containing UvrD helicase C terminal domain, consisting of remaining part of motif IV and motifs IVa–IVc and 161 amino acids of intervening sequence between motif IV and V, possess ssDNA-dependent ATPase and DNA helicase activities in vitro. Using immunodepletion assays we show that the ATPase and helicase activities are attributable to PfUDN and PfUDC1 proteins. The helicase activity can utilize the hydrolysis of all the nucleotide and deoxynucleotide triphosphates and the direction of unwinding is 3′ to 5′. The endogenous P. falciparum UvrD contains the characteristic DNA helicase activity. PfUDN interacts with PfMLH (P. falciparum MutL homologue) and modulates the endonuclease activity of PfMLH and PfMLH positively regulates the unwinding activity of PfUDN. We show that PfUvrD is expressed in the nucleus distinctly in the schizont stages of the intraerythrocytic development of the parasite and it colocalizes with PfMLH. These studies will make an important contribution in understanding the nucleic acid transaction in the malaria parasite.

疟疾是一种全球性疾病，亦是重大公共卫生难题。由于耐药性问题日益严峻，疟疾防控工作面临艰巨挑战。因此，亟需鉴定并表征新型的寄生虫特异性药物靶点。本研究针对恶性疟原虫（Plasmodium falciparum）来源的寄生虫特异性UvrD解旋酶（UvrD helicase），报道了其生化表征结果。该解旋酶包含两个功能片段：含UvrD解旋酶结构域的N端片段（PfUDN），其结构域涵盖解旋酶基序Q、Ia–Id、II、III以及大部分基序IV；以及含UvrD解旋酶C端结构域的C端片段（PfUDC1），其结构域包含基序IV的剩余区段、基序IVa–IVc，以及位于基序IV与V之间的161个氨基酸插入序列。二者在体外均具备单链DNA（single-stranded DNA, ssDNA）依赖的ATP酶（ATPase）活性与DNA解旋酶活性。通过免疫耗竭实验（immunodepletion assays），我们证实上述ATP酶与解旋酶活性均由PfUDN与PfUDC1蛋白介导。该解旋酶可利用所有核苷三磷酸与脱氧核苷三磷酸的水解供能，其DNA解旋方向为3′→5′。内源性恶性疟原虫UvrD同样具备典型的DNA解旋酶活性。PfUDN可与PfMLH（恶性疟原虫MutL同源蛋白）相互结合，并调控PfMLH的核酸内切酶活性；反之，PfMLH可正向调节PfUDN的解旋酶活性。研究发现，PfUvrD在疟原虫红细胞内发育周期的裂殖体阶段特异性表达于细胞核内，且与PfMLH存在共定位现象。本系列研究将为阐明疟疾寄生虫的核酸代谢机制提供重要理论支撑。