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

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

List of oligonucleotides.

<p>Primers 1–5 were used to isolate and modify the cDNA encoding <i>E</i>. <i>histolytica DMC1</i> and <i>RAD51</i>. H3, OL83-1, and OL90 were ³²P-radiolabeled using [³²P-<b>γ</b>]-ATP and T4-PNK. ³²P-H3 and ³²P-OL83-1 were annealed with H3c and OL83-2 oligonucleotides, respectively, to form double-stranded DNA substrates. ³²P-OL90 was used in the D-loop and nuclease protection assay.</p><p>List of oligonucleotides.</p

<i>eh</i>Dmc1 catalyzes D-loop formation.

<p><b>A.</b> Schematic of D-loop formation assay (ss, single-strand oligonucleotide; sc, supercoiled dsDNA). <b>B.</b><i>eh</i>Dmc1 was incubated with ³²P-radiolabeled OL90 ssDNA (lane 2), dsDNA (lane 3) prior to the addition of dsDNA or ssDNA (lanes 2 and 3, respectively), or both ssDNA and dsDNA (lane 4) simultaneously. Lane 1 is devoid of protein. After a 12 min incubation, an aliquot was removed and deproteinized prior to separation on an agarose gel. The mean percent of six independent experiments was graphed. Error bars represent SEM. <b>C.</b><i>eh</i>Dmc1 was incubated with ³²P-OL90 ssDNA in the presence of 2 mM nucleotide (ATP, lanes 1–4), ATP-<b>γ</b>-S (lane 5), ADP (lane 6) and AMP-PNP (lane 7). Lane 8 was devoid of nucleotide. At the indicated times, an aliquot was removed and processed as described in <b>B</b>. The mean percent of six independent experiments was graphed. Error bars represent SEM.</p

Stimulation of <i>eh</i>Dmc1-mediated DNA strand exchange activity by mHop2-Mnd1 and Ca²⁺.

<p><b>A.</b> Schematic of oligonucleotide strand exchange assay. <b>B.</b> A time course analysis of <i>eh</i>Dmc1 strand exchange activity (top panel) in the presence of 2 mM calcium (Ca²⁺), mHop2-Mnd1 (H2M1), and the combination of calcium and mHop2-Mnd1 (Ca²⁺ H2M1), as indicated. At the indicated times, an aliquot was removed and deproteinized. The reaction products were separated on 12% native polyacrylamide gels, and the gels were analyzed by a phosphorimager. Lane 1 is devoid of protein (Bl.). <b>C.</b> Mean values from three individual experiments from <b>B</b> were graphed. Error bars represent SEM. <b>D.</b> A 5 min time course analysis of <i>eh</i>Dmc1 strand exchange activity in the presence of mHop2-Mnd1 (H2M1) or the combination of 2 mM calcium and mHop2-Mnd1 (Ca²⁺ H2M1), as indicated. At the indicated times, an aliquot was removed, deproteinized and processed as described in <b>B</b>. Lane 1 (Bl.) is devoid of protein. <b>E.</b> Mean values of three independent experiments from <b>D</b> were plotted. Error bars represent SEM.</p

The <i>eh</i>Dmc1 nucleoprotein filament protects ssDNA in the presence of DNase I.

<p><b>A.</b>³²P-radiolabeled OL90 ssDNA was incubated with <i>eh</i>Dmc1 prior to the addition of DNase I. At the indicated times, an aliquot was removed and deproteinized. The reaction products were separated on a 12% native polyacrylamide gel followed by analysis with a phosphorimager. The mean percent protection of the ssDNA from DNase I digestion of three independent experiments was graphed. Error bars represent SEM. Lane 5 is devoid of protein. <b>B.</b>³²P-OL90 ssDNA was incubated with <i>eh</i>Dmc1 in the presence of 2.5 mM nucleotide (ATP, lane 1; ATP-γ-S, lane 2; ADP, lane 3; and AMP-PNP, lane 4) prior to the addition of DNase I. Lane 5 is devoid of protein and DNase I. Lane 6 is devoid of protein but contains DNase I. After a 10 min incubation, an aliquot was removed and processed as described in <b>A</b>. The mean percent protection of three independent experiments was graphed. Error bars represent SEM. (ss), ³²P-radiolabeled single-stranded OL90; (deg) degradation.</p

<i>eh</i>Dmc1 mediates plasmid length DNA strand exchange.

<p><b>A.</b> Schematic of the 3-strand homologous DNA pairing and strand exchange reaction. Homologous DNA pairing between the circular ssDNA (css) and linear dsDNA (lds) first forms a DNA joint molecule (jm). DNA strand exchange converts the joint molecule into a nicked circular duplex (nc) displacing the linear ssDNA (lss). <b>B.</b><i>eh</i>Dmc1 (12.5 μM) was incubated with ϕX174 virion ssDNA (css) to allow presynaptic filament formation to occur before the addition of hRPA (3.8 μM) and KCl (150 mM final concentration). The reaction was initiated by the addition of linearized double-strand ϕX174 DNA (lds) and spermidine. At the indicated time points, the reactions were deproteinized, subjected to agarose gel electrophoresis, and stained with ethidium bromide.</p

Characterization of the recombination activities of the \u3ci\u3eEntamoeba histolytica\u3c/i\u3e Rad51 recombinase

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

mHop2-Mnd1 interacts with <i>eh</i>Dmc1.

<p><i>eh</i>Dmc1 was mixed with Affi-Gel matrix conjugated to either mHop2-Mnd1 (lanes 2–4) or bovine serum albumin (BSA, lanes 5–7). After a wash, bound protein was eluted with SDS. The supernatant (S), wash (W), and eluate (E) were subjected to SDS-PAGE, and the gel was stained with Coomassie blue.</p

mHop2-Mnd1 and Ca²⁺ stimulate <i>eh</i>Dmc1-mediated D-loop formation.

<p><i>eh</i>Dmc1 was incubated with ³²P-radiolabeled OL90 ssDNA in the absence (lanes 1–4 and 9–12) or presence of calcium (lanes 5–8 and 13–16) and/or mHop2-Mnd1 (lanes 9–16). The reaction was initiated with the addition of supercoiled dsDNA. Aliquots were removed at the indicated times, deproteinized, and the reaction products were separated by agarose gel electrophoresis. Lanes 1, 5, 9, and 13 were lacking <i>eh</i>Dmc1. Mean values from three individual experiments were graphed. Error bars represent SEM.</p