Homologous Recombination in Protozoan Parasites and Recombinase Inhibitors

Abstract

Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that utilizes a homologous template to fully repair the damaged DNA. HR is critical to maintain genome stability and to ensure genetic diversity during meiosis. A specialized class of enzymes known as recombinases facilitate the exchange of genetic information between sister chromatids or homologous chromosomes with the help of numerous protein accessory factors. The majority of the HR machinery is highly conserved among eukaryotes. In many protozoan parasites, HR is an essential DSB repair pathway that allows these organisms to adapt to environmental conditions and evade host immune systems through genetic recombination. Therefore, small molecule inhibitors, capable of disrupting HR in protozoan parasites, represent potential therapeutic options. A number of small molecule inhibitors were identified that disrupt the activities of the human recombinase RAD51. Recent studies have examined the effect of two of these molecules on the Entamoeba recombinases. Here, we discuss the current understandings of HR in the protozoan parasites Trypanosoma, Leishmania, Plasmodium, and Entamoeba, and we review the small molecule inhibitors known to disrupt human RAD51 activity.

Exploiting CRISPR–Cas9 technology to investigate individual histone modifications

“…However, although the length of the target sequence should be sufficient to ensure targeted cleavage even in large genomes (∼3Gb), numerous studies have reported that Cas9 can induce off-target mutations, even at sequences that differ by 5 nt from the targeted site ( 11 , 39 , 40 ). Given the absence of an NHEJ pathway in T. brucei ( 13 ) and findings using the meganuclease I- SceI in T. brucei ( 14 ), we expect that cleavage at off-target sites, for which no repair template is provided, will not be repaired efficiently and will result in cell death rather than in unintended editing events. Nevertheless, to assess the degree of off-target mutations in T. brucei , we sequenced gDNA from wild type cells and a population of cells edited using the sgRNA-episome(SCD6-GFP.1) to 21.3x and 22.9x coverage, respectively.…”

“…Thus, while Cas9 has been successfully used to disrupt multicopy genes via repair through NHEJ, it has not been used for the precise editing of large multicopy gene arrays. Many protozoa, including the medically relevant parasites Trypanosoma brucei and Trypanosoma cruzi , the causative agents of sleeping sickness and Chagas disease, have very inefficient NHEJ and microhomology-mediated pathways ( 13 ). For T. brucei it was observed that in the absence of a repair template the majority of DSBs will result in cell death rather than repair by NHEJ or microhomology ( 14 ).…”

Apicomplexa and Histone Variants: What’s New?

“…DSB can be repaired by two main mechanisms: nonhomologous end joining (NHEJ) and homologous recombination repair (HRR); the first is an error-prone mechanism available along the cell cycle, and the second is an error-free mechanism active at S/G2 phases of cell cycle because of the requirement of sister chromatid as template [131][132][133][134]. Both mechanisms were described in T. gondii [14,135], but Plasmodium genus is thought to rely only on HRR [136][137][138].…”

Babesia bovis Rad51 ortholog influences switching of ves genes but is not essential for segmental gene conversion in antigenic variation

“…This phenomenon, in which short DNA patches are duplicated from a donor to a recipient gene, typically occurs without modification of the donor and at least superficially resembles HR-mediated DNA repair. DNA repair in apicomplexan and other protozoal parasites overall is not well understood [35,36]. It is even difficult to predict from the parasites' proteomes which repair pathways may be active.…”