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

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

<i>eh</i>Dmc1 binds DNA.

<p><b>A.</b> Increasing concentrations of <i>eh</i>Dmc1 (1.3 μM, lane 2; 2.6 μM, lane 3; 3.9 μM, lane 4; and 5.2 μM, lane 5) were incubated with ssDNA (³²P-labeled H3 ssDNA). <b>B.</b> The mean binding percentages were graphed for three independent experiments from <b>A</b>. Error bars represent SEM. <b>C.</b> Increasing concentrations of <i>eh</i>Dmc1 (5.2 μM, lane 2; 10.4 μM, lane 3; 20.8 μM, lane 4; and 31.2 μM, lane 5) were incubated with dsDNA (³²P-labeled H3 annealed to H3c). <b>D.</b> The mean binding percentages were graphed for three independent experiments from <b>C</b>. Error bars represent SEM. Lane 1 for <b>A</b> and <b>C</b> is devoid of protein, and lane 6 for <b>A</b> and <b>C</b> was SDS/PK (S/P) treated containing the highest concentration of <i>eh</i>Dmc1.</p

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

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

<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