Nucleotide excision repair in Trypanosoma brucei: specialization of transcription‐coupled repair due to multigenic transcription

Abstract

Nucleotide excision repair (NER) is a highly conserved genome repair pathway acting on helix distorting DNA lesions. NER is divided into two subpathways: global genome NER (GG-NER), which is responsible for repair throughout genomes, and transcription-coupled NER (TC-NER), which acts on lesions that impede transcription. The extent of the Trypanosoma brucei genome that is transcribed is highly unusual, since most genes are organized in multigene transcription units, each transcribed from a single promoter. Given this transcription organization, we have addressed the importance of NER to T. brucei genome maintenance by performing RNAi against all predicted contributing factors. Our results indicate that TC-NER is the main pathway of NER repair, but only CSB, XPBz and XPG contribute. Moreover, we show that UV lesions are inefficiently repaired in T. brucei, perhaps due to preferential use of RNA polymerase translesion synthesis. RNAi of XPC and DDB was found to be lethal, and we show that these factors act in inter-strand cross-link repair. XPD and XPB appear only to act in transcription, not repair. This work indicates that the predominance of multigenic transcription in T. brucei has resulted in pronounced adaptation of NER relative to the host and may be an attractive drug target.

Deciphering the interstrand crosslink DNA repair network expressed byTrypanosoma brucei

“…To evaluate whether deletion of Tb csb , Tb mre11 , Tb exo1 , Tb rad5 or Tbrev3 from the T. brucei genome altered the parasites sensitivity to DNA damage, the null mutants were grown in the presence of hydroxyurea, phleomycin or methyl methanesulfonate, or cultured following exposure to a single dose of UV, and the EC 50 values towards each treatment determined (Table 1; S2C & D Figs). Cells lacking TbCSB were 5-fold more susceptible to UV than wild type, in keeping with previous observations [41], while parasites lacking TbEXO1 displayed an increased sensitivity (2-fold) towards phleomycin. In keeping with the published literature, TbMRE11 deficient parasites were more susceptible to phleomycin (12-fold) and methyl methanesulfonate (2.5-fold), agents that promote DSBs (Table 1: S2C & D Figs) [46, 47].…”

“…Using reciprocal BLAST the T. brucei genes encoding for homologues of these enzymes plus their flanking regions were identified from the TriTrypDB (http://tritrypdb.org/tritrypdb/) and NCBI (https://www.ncbi.nlm.nih.gov/) databases. For TbMRE11 and TbCSB, the sequences arising from these searches corresponded to those previously reported [41, 46, 47]. In contrast, the genes described here as encoding EXO1, RAD5 and REV3 orthologues have not been characterised.…”

“…To determine the extent of this system in T. brucei , FA proteins (complete or specific domains) from humans, Danio rerio, Drosophila melanogaster, Arabidopsis thaliana and S. cerevisiae were used to search for trypanosomal homologues held on the TriTryp and NCBI databases. Out of the 26 sequences examined, T. brucei possessed discernible homologues for 7 including BRCA2 (FANCD1), RAD51 (FANCR), RAD51C (FANCO) and XPF (FANCQ), enzymes that play a role in this parasite’s HR and NER pathways (Fig 1D) [41, 50–52]. All of the sequences identified were so-called FA effector proteins with none corresponding to components of the FA core complex (FANCA, −B, −C, −E, −F, −G, −L, −M and −T), recruitment factors (FANCD2A and −I) or FA associate protein (FAAP10, −16, −20, −24 and −100).…”

“…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.…”

Neglected Tropical Diseases in the Post-Genomic Era

“…Their mono-allelic and sequential expression on the parasite surface drives the process of 'antigenic variation' which is essential for survival in the bloodstream of mammalian hosts. the identification of missing or divergent pathways involved in DNA repair and replication facilitated by analysis of the genome sequences of trypanosomatids [13] promises to provide novel drug targets [29].…”