Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

Abstract

The protozoan parasite responsible for human amoebiasis is Entamoeba histolytica. An important facet of the life cycle of E. histolytica involves the conversion of the mature trophozoite to a cyst. This transition is thought to involve homologous recombination (HR), which is dependent upon the Rad51 recombinase. Here, a biochemical characterization of highly purified ehRad51 protein is presented. The ehRad51 protein preferentially binds ssDNA, forms a presynaptic filament and possesses ATP hydrolysis activity that is stimulated by the presence of DNA. Evidence is provided that ehRad51 catalyzes robust DNA strand exchange over at least 5.4 kilobase pairs. Although the homologous DNA pairing activity of ehRad51 is weak, it is strongly enhanced by the presence of two HR accessory cofactors, calcium and Hop2-Mnd1. The biochemical system described herein was used to demonstrate the potential for targeting ehRad51 with two small molecule inhibitors of human RAD51. We show that 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) inhibited ehRad51 by interfering with DNA binding and attenuated encystation in Entamoeba invadens, while B02 had no effect on ehRad51 strand exchange activity. These results provide insight into the underlying mechanism of homology-directed DNA repair in E. histolytica.

Flow cytometric characterization of encystation in Entamoeba invadens

“…DIDS is a commercially-available small molecule inhibitor of human Rad51 [19], a DNA recombinase that regulates homology search and strand exchange during homologous recombination. We have previously shown that DIDS decreases the activity of eh Rad51, the Entamoeba ortholog of Rad51, and inhibits encystation [20]. E. invadens cells were induced to encyst in the absence or presence of DIDS (30 µM or 150 µM) for 48 h. Manual counting of detergent-resistant cells confirmed that DIDS inhibits terminal differentiation (Fig.…”

“…Trophozoites (Control) were induced to encyst for 48 h in the presence of Gal (10 mM [6]), GlcNAc (10 mM [6]), GalNAc (10 mM [6]) or DIDS (30 µM or 150 µM [20]). Cells or DRCs were stained and analyzed as described in Fig.…”

Data on Rad51 amino acid sequences from higher and lower eukaryotic model organisms and parasites

“…The data described, include supporting information on sequence conservation and identity of Rad51 for the analysis by Kelso et al, in press [1]. A Rad51 amino acid sequence alignment from nineteen different vertebrate and invertebrate organisms is shown.…”

“…3). A comparison of Entamoeba histolytica Rad51 to the other species was analyzed in the previously mentioned article [1] and by Lopez-Casamichana et al, 2008 [4].…”

Homologous Recombination in Protozoan Parasites and Recombinase Inhibitors

“…Biochemically, Lopez-Casamichana et al (2008) showed that partially purified Eh Rad51 bound DNA and facilitated homologous DNA pairing ( Lopez-Casamichana et al, 2008 ). The findings were recently extended by a report that demonstrated Eh Rad51 was not only capable of binding DNA and forming D-loops, but that Eh Rad51 hydrolyzed ATP, formed presynaptic filaments, and catalyzed DNA strand exchange over thousands of base pairs ( Kelso et al, 2016 ). Although E. histolytica reproduce asexually through binary fission, the occurrence of meiosis in E. histolytica was first proposed to occur during encystation based on increased expression of meiosis-specific genes ( Ehrenkaufer et al, 2013 ).…”