Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

Abstract

Meiosis depends on homologous recombination (HR) in most sexually reproducing organisms. Efficient meiotic HR requires the activity of the meiosis-specific recombinase, Dmc1. Previous work shows Dmc1 is expressed in Entamoeba histolytica, a eukaryotic parasite responsible for amoebiasis throughout the world, suggesting this organism undergoes meiosis. Here, we demonstrate Dmc1 protein is expressed in E. histolytica. We show that purified ehDmc1 forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. The DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. In combination, calcium and Hop2-Mnd1 dramatically increase the rate of DNA strand exchange activity of ehDmc1. The biochemical system described herein provides a basis on which to better understand the role of ehDmc1 and other HR proteins in E. histolytica.

Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

“…We first cloned the RAD51 gene from the E. histolytica genome. The sequence encoding the 6 histidine epitope tag was fused to the 5′ end of the ehRAD51 gene by PCR and was inserted into a bacterial expression plasmid to be used for protein production [37]. Using a combination of affinity and ion exchange chromatography, eh Rad51 was purified to near homogeneity (Fig.…”

“…The isolation and modification of the E. histolytica Rad51 gene (XM 648984) to create the pET- ehRAD51 -(HIS) 6 bacterial expression plasmid was previously described [37]. …”

“…The purification of eh Rad51 was performed as previously described [37] with modifications. Briefly, eh Rad51 was expressed in the BL21 (Novagen) strain of E. coli cells.…”

“…Existing protocols were used to express and purify E. histolytica Dmc1 ( eh Dmc1) [37], murine Hop2-Mnd1 [40], human RAD51, and human RPA [41]. …”

“…To determine the interaction of eh Rad51 with mHop2-Mnd1, a pull-down was performed as described [37]. Briefly, mHop2-Mnd1 (3.5 mg) and BSA (5 mg) were immobilized on 0.5 ml of Affi-gel 15 matrix (BioRad), separately, according to the manufacturer’s instructions.…”