Abstract
Trypanosoma brucei is a unicellular parasite responsible for African trypanosomiasis or sleeping sickness. It contains a single PARP enzyme opposed to many higher eukaryotes, which have numerous PARPs. PARPs are responsible for a post-translational modification, ADP-ribosylation, regulating a multitude of cellular events. T. brucei PARP, like human PARPs-1-3, is activated by DNA binding and it potentially functions in DNA repair processes. Here we characterized activation requirements, structure and subcellular localization of T. brucei PARP. T. brucei PARP was found to be selectively activated by 5′ phosphorylated and 3′ phosphorylated DNA breaks. Importantly, the N-terminal region is responsible for high-affinity DNA-binding and required for DNA-dependent enzymatic activation. This module is also required for nuclear localization of the protein in response to oxidative stress. Solution structures of activating and non-activating PARP-DNA complexes were determined with small-angle X-ray scattering revealing distinct differences in their DNA-binding modes.
Highlights
Trypanosoma brucei is a unicellular parasitic protozoa responsible for African trypanosomiasis or sleeping sickness, a scourge in Sub-Saharan Africa for both livestock and human health
We have previously shown that TbPARP is catalytically activated by damaged DNA and it most likely functions in DNA-damage response and DNA-repair like hPARPs-1-36
While hPARP-1 utilizes three zinc-fingers for DNA binding, hPARPs-2 and 3 have a positively charged N-terminus that is utilized for DNA interaction
Summary
Trypanosoma brucei is a unicellular parasitic protozoa responsible for African trypanosomiasis or sleeping sickness, a scourge in Sub-Saharan Africa for both livestock and human health. The parasite contains only a single PARP enzyme (TbPARP), which has a similar domain organization as hPARPs-2 and 3, including the WGR, RD, and ARTD domains (Fig. 1). It contains a positively charged N-terminus, with higher sequence similarity to hPARP-2 than to hPARP-3. Addition, inhibition of the catalytic activity of TbPARP did not inhibit the growth of T. brucei procyclic and bloodstream forms[6]. These results are in contrast to what have been observed for the related parasite T. cruzi[7]. Our results indicate a high degree of substrate selectivity in the DNA activation process and identify the N-terminus as a key domain in DNA-binding, DNA-dependent catalytic activation, and nuclear localization
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