Background: Hed1 attenuates the activity of Rad51 during meiotic recombination to allow Dmc1-dependent formation of interhomolog crossovers. Results: Hed1 possesses DNA binding and self-association activities and stabilizes Rad51 presynaptic filament. Conclusion: DNA binding, Rad51 interaction, and self-association are essential for Hed1 function as a key regulator of meiotic recombination. Significance: Our results shed light on the mechanism of interhomolog bias in meiotic recombination.
Meiotic homologous recombination in Saccharomyces cerevisiae involves formation of nucleoprotein filaments of Rad51 and Dmc1 that mediate DNA strand exchange between homologous chromosomes. The Mei5-Sae3 protein complex functions as a recombination mediator to promote nucleation of the Dmc1 recombinase onto replication protein A-coated single-stranded DNA. Here, we have expressed and purified the Mei5 protein, Sae3 protein and the Mei5-Sae3 complex for biochemical studies. We show the Mei5-Sae3 complex preferentially binds a fork-like DNA substrate to 3′ overhanging DNA, single-stranded DNA or double-stranded DNA. We demonstrate that Mei5 confers DNA binding activity to the Mei5-Sae3 complex. We determined Mei5-Sae3 interacts with the Rad51 recombinase through the N-terminal domain of Mei5. Unlike Rad52, Mei5-Sae3 lacks recombination mediator activity for Rad51. Importantly, we find that the Mei5-Sae3 complex does not harbor single-strand DNA annealing activity. These properties of the Mei5-Sae3 complex distinguishes it from the Rad52 protein, which serves as the mediator of Rad51 and is involved in the single-strand DNA annealing pathway of homologous recombination.
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.
During meiosis, the RAD51 recombinase and its meiosis-specific homolog DMC1 mediate DNA strand exchange between homologous chromosomes. The proteins form a right-handed nucleoprotein complex on ssDNA called the presynaptic filament. In an ATP-dependent manner, the presynaptic filament searches for homology to form a physical connection with the homologous chromosome. We constructed two variants of hDMC1 altering the conserved lysine residue of the Walker A motif to arginine (hDMC1K132R) or alanine (hDMC1K132A). The hDMC1 variants were expressed in Escherichia coli and purified to near homogeneity. Both hDMC1K132R and hDMC1K132A variants were devoid of ATP hydrolysis. The hDMC1K132R variant was attenuated for ATP binding that was partially restored by the addition of either ssDNA or calcium. The hDMC1K132R variant was partially capable of homologous DNA pairing and strand exchange in the presence of calcium and protecting DNA from a nuclease, while the hDMC1K132A variant was inactive. These results suggest that the conserved lysine of the Walker A motif in hDMC1 plays a key role in ATP binding. Furthermore, the binding of calcium and ssDNA promotes a conformational change in the ATP binding pocket of hDMC1 that promotes ATP binding. Our results provide evidence that the conserved lysine in the Walker A motif of hDMC1 is critical for ATP binding which is required for presynaptic filament formation.
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.
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