Unravelling the role of SNM1 in the DNA repair system of Trypanosoma brucei

Abstract

SummaryAll living cells are subject to agents that promote DNA damage. A particularly lethal lesion are interstrand cross-links (ICL), a property exploited by several anticancer chemotherapies. In yeast and humans, an enzyme that plays a key role in repairing such damage are the PSO2/SNM1 nucleases. Here, we report that Trypanosoma brucei, the causative agent of African trypanosomiasis, possesses a bona fide member of this family (called TbSNM1) with expression of the parasite enzyme able to suppress the sensitivity yeast pso2Δ mutants display towards mechlorethamine, an ICL-inducing compound. By disrupting the Tbsnm1 gene, we demonstrate that TbSNM1 activity is nonessential to the medically relevant T. brucei life cycle stage. However, trypanosomes lacking this enzyme are more susceptible to bi-and tri-functional DNA alkylating agents with this phenotype readily complemented by ectopic expression of Tbsnm1. Genetically modified variants of the null mutant line were subsequently used to establish the anti-parasitic mechanism of action of nitrobenzylphosphoramide mustard and aziridinyl nitrobenzamide prodrugs, compounds previously shown to possess potent trypanocidal properties while exhibiting limited toxicity to mammalian cells. This established that these agents, following activation by a parasite specific type I nitroreductase, produce metabolites that promote formation of ICLs leading to inhibition of trypanosomal growth.

Deciphering the interstrand crosslink DNA repair network expressed byTrypanosoma brucei

“…The resultant data was plotted as dose response curves from which EC 50 values were extrapolated (Fig 3). Previous work has shown that TbSNM1 plays an important role in resolving the damage caused by mechlorethamine with our data confirming this earlier finding [42]: Cells lacking TbSNM1 are >8-fold more susceptible to this compound as compared to controls. A similar alteration in sensitivity was also observed in cells deficient in TbMRE11, TbCSB or TbEXO1 although the difference in EC 50 values displayed by these 3 mutants relative to wild type was not as great as that noted for Tb snm1 Δ cells; Tb mre11 Δ, Tb csb Δ and Tb exo1 Δ cells were 3-, 4- and 2-fold more susceptible to mechlorethamine than wild type, respectively (Figs 3A-C) This could suggest the relative importance of each enzyme in T. brucei ’s so-called ICL REPAIRtoire.…”

“…Additionally, TbRAD5 and TbREV3 although not primarily involved in resolving ICLs, were also taken forward to determine whether their activities in this repair network becomes apparent in the absence of TbSNM1: The role of some yeast factors in ICL repair are only apparent when the activity of another DNA repair protein is missing [63]. To generate the double null mutant lines, the hyg-and neo -based integration vectors for each targeted gene were linearised and sequentially transformed into T. brucei Tb snm1 Δ cells (in this mutant line, Tbsnm1 has been disrupted using vectors based around resistance cassettes that includes the gene encoding for blasticidin-S-deaminase (bla) or puromycin N-acetyltransferase ( pac ) [42]). Following selection, all putative double null clones were validated using the PCR-based strategies outlined previously with this confirming that the introduced DNA fragments had integrated into the T. brucei genome and that the recombinant parasites were no longer expressing both disrupted genes (Fig 2).…”

“…In many cases, the use of null mutant lines has provided the primary route for the identification of which DNA repair factors function in an organism’s ICL REPAIRtoire and in deciphering how these components interact [45, 63, 71, 72]. Transferring this approach to T. brucei , lines lacking a single potential ICL repair factor were made (TbCSB, TbEXO1, TbMRE11, TbRAD5 or TbREV3) or sourced (TbSNM1, TbRAD51 or TbBRCA2 [42, 50, 52]) with these subsequently phenotyped using mechlorethamine as a selective agent. For most parasite lines tested, an increased susceptibility to the ICL-inducing agent was observed, demonstrating that TbSNM1, TbCSB, TbEXO1, TbMRE11, TbRAD51 and TbBRCA2 all contribute to the trypanosomal ICL REPAIRtoire.…”

“…014 % (v/v) β-mercaptoethanol and 10 % (v/v) foetal bovine serum (FBS) at 37 °C under a 5 % (v/v) CO2 atmosphere [80]. Derivatives of the ‘221’ line null for DNA repair enzymes (S1 Table), including cells lacking Tbsnm1, Tbrad51 and Tbbrca2 [42, 50, 52], were maintained in the above growth medium containing 2.5 μg ml −1 hygromycin, 10 μg ml −1 blasticidin, 2 μg ml −1 puromycin and/or 2 μg ml −1 G418.…”

“…Some components of the global genome NER (GG-NER) pathway (e.g. TbXPC and TbDDB) may potentially be involved in this process [41] while T. brucei cells null for TbSNM1, a member of the SNM1/PSO2 family of nucleases, display sensitivity only to ICL-inducing compounds [42]. Here, using a classical genetics-based approach, we analyse whether other DNA repair enzymes from the parasite’s transcription-coupled NER (TC-NER) (TbCSB), HR (TbMRE11, TbRAD51, TbBRCA2 and TbEXO1), mismatch repair (MMR; TbEXO1) and DNA damage tolerance (TbREV3 and TbRAD5) pathways contribute to the trypanosomal ICL repair network then assess their interplay with TbSNM1 and with each other.…”