The parasites of the order kinetoplastidae including Leishmania spp. emerge from most ancient phylogenic branches of unicellular eukaryotic lineages. In their life cycle, topoisomerase I plays a significant role in carrying out vital cellular processes. Camptothecin (CPT), an inhibitor of DNA topoisomerase I, induces programmed cell death (PCD) both in the amastigotes and promastigotes form of L. donovani parasites. CPT-induced cellular dysfunction in L. donovani promastigotes is characterized by several cytoplasmic and nuclear features of apoptosis. CPT inhibits cellular respiration that results in mitochondrial hyperpolarization taking place by oligomycin-sensitive F0-F1 ATPase-like protein in leishmanial cells. During the early phase of activation, there is an increase in reactive oxygen species (ROS) inside cells, which causes subsequent elevation in the level of lipid peroxidation and decrease in reducing equivalents like GSH. Endogenous ROS formation and lipid peroxidation cause eventual loss of mitochondrial membrane potential. Furthermore, cytochrome c is released into the cytosol in a manner independent of involvement of CED3/CPP32 group of proteases and unlike mammalian cells it is insensitive to cyclosporin A. These events are followed by activation of both CED3/CPP32 and ICE group of proteases in PCD of Leishmania. Taken together, our study indicates that different biochemical events leading to apoptosis in leishmanial cells provide information that could be exploited to develop newer potential therapeutic targets.

Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3′-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1–PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1.