The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions

Abstract

In Trypanosoma cruzi, the etiologic agent of Chagas disease, Rad51 (TcRad51) is a central enzyme for homologous recombination. Here we describe the different roles of TcRad51 in DNA repair. Epimastigotes of T. cruzi overexpressing TcRAD51 presented abundant TcRad51-labeled foci before gamma irradiation treatment, and a faster growth recovery when compared to single-knockout epimastigotes for RAD51. Overexpression of RAD51 also promoted increased resistance against hydrogen peroxide treatment, while the single-knockout epimastigotes for RAD51 exhibited increased sensitivity to this oxidant agent, which indicates a role for this gene in the repair of DNA oxidative lesions. In contrast, TcRad51 was not involved in the repair of crosslink lesions promoted by UV light and cisplatin treatment. Also, RAD51 single-knockout epimastigotes showed a similar growth rate to that exhibited by wild-type ones after treatment with hydroxyurea, but an increased sensitivity to methyl methane sulfonate. Besides its role in epimastigotes, TcRad51 is also important during mammalian infection, as shown by increased detection of T. cruzi cells overexpressing RAD51, and decreased detection of single-knockout cells for RAD51, in both fibroblasts and macrophages infected with amastigotes. Besides that, RAD51-overexpressing parasites infecting mice also presented increased infectivity and higher resistance against benznidazole. We thus show that TcRad51 is involved in the repair of DNA double strands breaks and oxidative lesions in two different T. cruzi developmental stages, possibly playing an important role in the infectivity of this parasite.

The Influence of Recombinational Processes to Induce Dormancy in Trypanosoma cruzi

“…Sánchez-Valdéz et al (2018) hypothesized that homologous recombination is the cause for dormancy in T. cruzi. It is well-known that the recombination pivot is TcRad51, a protein responsible for the repair of DSBs-either induced by high levels of gamma radiation, or caused by other genotoxic agents that lead to transcriptional and replicational fork arrest (Regis-da-Silva et al, 2006;Gomes Passos Silva et al, 2018). We then observed the effect of gamma radiation in inducing arrest in T. cruzi epimastigotes.…”

“…Gamma radiation generates DSBs in DNA, which are repaired by homologous recombination in T. cruzi. We have previously shown that epimastigotes exposed to 500 Gy of gamma radiation enter in a cell cycle arrest for 4 days, by the end of which are able to resume their cellular growth (Gomes Passos Silva et al, 2018). In order to analyze in detail how gamma radiation affects epimastigotes of T. cruzi from CL Brener strain, we irradiated cells with 500 Gy of gamma rays and labeled them immediately with CFSE.…”

“…Since increased TcRAD51 transcription levels are directly related to higher dormancy in T. cruzi (Figure 6), we hypothesized that the CL Brener RAD51+/− mutant, which harbors only one copy of TcRAD51 (Gomes Passos Silva et al, 2018), would exhibit a decreased percentage of arrested parasites when compared to CL Brener WT . In fact, epimastigote cultures of CL Brener RAD51+/− showed a decreased percentage of cells presenting high CFSE fluorescence intensity, indicating a high duplication rate and a reduced percentage of dormant cells when compared to CL Brener WT parasites (Figures 7B,C).…”

“…T. cruzi overexpressing TcRAD51 is capable of resolving DSBs in both epimastigotes and amastigotes. On the other hand, TcRAD51-deficient epimastigotes and amastigotes are unable to properly repair DSBs (Gomes Passos Silva et al, 2018). Homologous recombination and TcRad51 also play a relevant role in genetic exchange processes since the overexpression of TcRAD51 is capable of stabilizing hybrid T. cruzi cells.…”

RAD50 promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes

“…The persistence of RPA foci in the 1 HR rad50 cells is further evidence of defective repair in these cells, explained by RPA remaining associated with ssDNA at sites of damage. This observation may also reflect a damage tolerance strategy employed by T. brucei 22 , but in these circumstances γH2A and RAD51 are not recruited to the break after replication, unlike what is seen in T. cruzi where ionizing radiation tolerance is dependent on RAD51 67 . DNA resection following a DSB is one of the early steps in the DDR.…”