Sak1 kinase interacts with Pso2 nuclease in response to DNA damage induced by interstrand crosslink-inducing agents in Saccharomyces cerevisiae

Munari, Fernanda Mosena; Revers, L. F.; Cardone, Jacqueline Moraes; Immich, Bruna Frielink; Moura, Dinara Jaqueline; Guecheva, Temenouga Nikolova; Bonatto, Diego; Laurino, Jomar Pereira; Saffi, Jenifer; Brendel, Martin; Henriques, João Antônio Pêgas

doi:10.1016/j.jphotobiol.2013.11.024

Cited by 7 publications

(3 citation statements)

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“…Therefore, if formation of PSO2/SNM1‐containing DNA repair complexes at the site of ICL damage does involve PCNA ubiquitylation, then the molecular mechanisms underlying TbSNM1 recruitment to such lesions occurs through an as yet uncharacterised UIM or via interactions involving a conserved adapter protein. Recently, it has been shown that the β‐CASP domain of Pso2p can be phosphorylated leading to the suggestion that this event may play a role in modulating the enzyme's exo‐ or endo‐nucleolytic activity (Munari et al ., ). Whether TbSNM1 undergoes a similar post‐translational modification, and how this effects its nuclease activity has yet to be established.…”

Section: Discussionmentioning

confidence: 97%

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

Sullivan

Tong

Wong

et al. 2015

Molecular Microbiology

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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.

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Section: Discussionmentioning

confidence: 97%

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

Sullivan

Tong

Wong

et al. 2015

Molecular Microbiology

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“…Alternatively, or in addition, yet unidentified post-translational modification of Pso2 may regulate its intracellular distribution. Concordant with this idea, phosphorylation of Pso2 has been shown to modulate its exo- or endo-nucleolytic activity (Munari et al . 2014).…”

Section: Discussionmentioning

confidence: 77%

Dual targeting ofSaccharomyces cerevisiaePso2 to mitochondria and the nucleus, and its functional relevance in the repair of DNA interstrand crosslinks

Muniyappa

Somashekara

2022

Preprint

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Repair of DNA interstrand crosslinks (ICLs) involves a functional interplay among different DNA surveillance and repair pathways. Previous work has shown that ICL-inducing agents cause damage to Saccharomyces cerevisiae nuclear and mitochondrial DNA (mtDNA), and its pso2/snm1 mutants exhibit a petite phenotype followed by loss of mtDNA integrity and copy number. Complex as it is, the cause and underlying molecular mechanisms remains elusive. Here, by combining a wide range of approaches with in vitro and in vivo analyses, we assessed the subcellular localization and function of Pso2. We found evidence that the nuclear-encoded Pso2 contains one mitochondrial targeting sequence (MTS) and two nuclear localization signals (NLS1 and NLS2), although NLS1 resides within the MTS. Further analysis revealed that Pso2 is a dual-localized ICL repair protein; it can be imported into both nucleus and mitochondria, and that genotoxic agents enhance its abundance in the latter. While MTS is essential for mitochondrial Pso2 import, either NLS1 or NLS2 is sufficient for its nuclear import; this implies that the two NLS motifs are functionally redundant. Ablation of MTS abrogated mitochondrial Pso2 import, and concomitantly, raised its levels in the nucleus. Strikingly, mutational disruption of both NLS motifs blocked the nuclear Pso2 import; at the same time, they enhanced its translocation into the mitochondria, consistent with the notion that the relationship between MTS and NLS motifs is competitive. However, the nuclease activity of import-deficient species of Pso2 was not impaired. The potential relevance of dual-targeting of Pso2 into two DNA-bearing organelles is discussed.

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“…This situation resembles that noted in other organisms although there are some key differences. In S. cerevisiae , PSO2 (the yeast SNM1 homologue) displays a non-epistatic interaction with EXO1 and all tested DSB repair protein, including MRE11 [45, 72, 74, 75]. It also associates with several NER factors such as RAD1 (XPF), RAD3 (XPD), RAD4 (XPC) and RAD14 (XPA) [22, 72] although whether it interacts with RAD26 (the yeast CSB homologue) remains unclear [22].…”

Section: Discussionmentioning

confidence: 99%

Deciphering the interstrand crosslink DNA repair network expressed byTrypanosoma brucei

Dattani

Wilkinson

2019

Preprint

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17Interstrand crosslinks (ICLs) represent a highly toxic form of DNA damage that can block 18 essential biological processes including DNA replication and transcription. To combat their 19 deleterious effects all eukaryotes have developed cell cycle-dependent repair strategies that co-20 opt various factors from 'classical' DNA repair pathways to resolve such lesions. Here, we 21 report that Trypanosoma brucei, the causative agent of African trypanosomiasis, possesses 22 such systems that show some intriguing differences to those mechanisms expressed in other 23 organisms. Following the identification of trypanosomal homologues encoding for CSB, 24 EXO1, SNM1, MRE11, RAD51 and BRCA2, gene deletion coupled with phenotypic studies 25 demonstrated that all the above factors contribute to this pathogen's ICL REPAIRtoire with 26 their activities split across two epistatic groups. We show that one network, which encompasses 27 TbCSB, TbEXO1 and TbSNM1, may operate throughout the cell cycle to repair ICLs 28 encountered by transcriptional detection mechanisms while the other relies on homologous 29 recombination enzymes that together may resolve lesions responsible for the stalling of DNA 30 replication forks. By unravelling and comparing the T. brucei ICL REPAIRtoire to those 31 systems found in its host, targets amenable to inhibitor design may be identified and could be 32 used alongside trypanocidal ICL-inducing agents to exacerbate their effects. 33 34 Author summary 35 Parasites belonging to the Trypanosoma brucei complex cause a human and animal infections 36 collectively known as African trypanosomiasis. Drugs used against these diseases are 37 problematic as medical supervision is required for administration, they are costly, have limited 38 efficacy, may cause unwanted side effects while drug resistance is emerging. Against this 39 backdrop, there is a need for new therapies targeting these neglected tropical 40 diseases. Previous studies have shown compounds that induce DNA interstrand crosslinks 41 (ICLs) formation are effective trypanocidal agents with the most potent invariably functioning 42 as prodrugs. Despite the potential of ICL-inducing compounds to treat African trypanosomiasis 43 little is known about the ICL repair mechanisms expressed by trypanosomes. Using a 44 combination of gene deletion and epistatic analysis we report the first systematic dissection of 45 how ICL repair might operate in T. brucei, a diverged eukaryote. It sheds light on the 46 conservation and divergence of ICL repair in one of only a handful of protists that can be 47 studied genetically, and offers the promise of developing or exploiting ICL-causing agents as 48 3 new anti-parasite therapies. These findings emphasise the novelty and importance of 49 understanding ICL repair in T. brucei and, more widely, in non-model eukaryotes.50 51 52 Spread by the hematophagous feeding behaviour of tsetse flies, protozoan parasites 53 belonging to the Trypanosoma brucei species complex are responsible for a group of human 54 and anim...

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Sak1 kinase interacts with Pso2 nuclease in response to DNA damage induced by interstrand crosslink-inducing agents in Saccharomyces cerevisiae

Cited by 7 publications

References 80 publications

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

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

Dual targeting ofSaccharomyces cerevisiaePso2 to mitochondria and the nucleus, and its functional relevance in the repair of DNA interstrand crosslinks

Deciphering the interstrand crosslink DNA repair network expressed byTrypanosoma brucei

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