A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments

filament

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

The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing

Homologous recombination (HR) is a template-driven repair pathway that mends DNA double-stranded breaks (DSBs), and thus helps to maintain genome stability. The RAD51 recombinase facilitates DNA joint formation during HR, but to accomplish this task, RAD51 must be loaded onto the single-stranded DNA. DSS1, a candidate gene for split hand/split foot syndrome, provides the ability to recognize RPA-coated ssDNA to the tumor suppressor BRCA2, which is complexed with RAD51. Together BRCA2-DSS1 displace RPA and load RAD51 onto the ssDNA. In addition, the BRCA2 interacting protein BCCIP normally colocalizes with chromatin bound BRCA2, and upon DSB induction, RAD51 colocalizes with BRCA2-BCCIP foci. Down-regulation of BCCIP reduces DSB repair and disrupts BRCA2 and RAD51 foci formation. While BCCIP is known to interact with BRCA2, the relationship between BCCIP and RAD51 is not known. In this study, we investigated the biochemical role of the β-isoform of BCCIP in relation to the RAD51 recombinase. We demonstrate that BCCIPβ binds DNA and physically and functionally interacts with RAD51 to stimulate its homologous DNA pairing activity. Notably, this stimulatory effect is not the result of RAD51 nucleoprotein filament stabilization; rather, we demonstrate that BCCIPβ induces a conformational change within the RAD51 filament that promotes release of ADP to help maintain an active presynaptic filament. Our findings reveal a functional role for BCCIPβ as a RAD51 accessory factor in HR

Homologous Recombination in Protozoan Parasites and Recombinase Inhibitors

Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that utilizes a homologous template to fully repair the damaged DNA. HR is critical to maintain genome stability and to ensure genetic diversity during meiosis. A specialized class of enzymes known as recombinases facilitate the exchange of genetic information between sister chromatids or homologous chromosomes with the help of numerous protein accessory factors. The majority of the HR machinery is highly conserved among eukaryotes. In many protozoan parasites, HR is an essential DSB repair pathway that allows these organisms to adapt to environmental conditions and evade host immune systems through genetic recombination. Therefore, small molecule inhibitors, capable of disrupting HR in protozoan parasites, represent potential therapeutic options. A number of small molecule inhibitors were identified that disrupt the activities of the human recombinase RAD51. Recent studies have examined the effect of two of these molecules on the Entamoeba recombinases. Here, we discuss the current understandings of HR in the protozoan parasites Trypanosoma, Leishmania, Plasmodium, and Entamoeba, and we review the small molecule inhibitors known to disrupt human RAD51 activity

Homologous Recombination in Protozoan Parasites and Recombinase Inhibitors

Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that utilizes a homologous template to fully repair the damaged DNA. HR is critical to maintain genome stability and to ensure genetic diversity during meiosis. A specialized class of enzymes known as recombinases facilitate the exchange of genetic information between sister chromatids or homologous chromosomes with the help of numerous protein accessory factors. The majority of the HR machinery is highly conserved among eukaryotes. In many protozoan parasites, HR is an essential DSB repair pathway that allows these organisms to adapt to environmental conditions and evade host immune systems through genetic recombination. Therefore, small molecule inhibitors, capable of disrupting HR in protozoan parasites, represent potential therapeutic options. A number of small molecule inhibitors were identified that disrupt the activities of the human recombinase RAD51. Recent studies have examined the effect of two of these molecules on the Entamoeba recombinases. Here, we discuss the current understandings of HR in the protozoan parasites Trypanosoma, Leishmania, Plasmodium, and Entamoeba, and we review the small molecule inhibitors known to disrupt human RAD51 activity

Homologous Recombination in Protozoan Parasites and Recombinase Inhibitors

Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that utilizes a homologous template to fully repair the damaged DNA. HR is critical to maintain genome stability and to ensure genetic diversity during meiosis. A specialized class of enzymes known as recombinases facilitate the exchange of genetic information between sister chromatids or homologous chromosomes with the help of numerous protein accessory factors. The majority of the HR machinery is highly conserved among eukaryotes. In many protozoan parasites, HR is an essential DSB repair pathway that allows these organisms to adapt to environmental conditions and evade host immune systems through genetic recombination. Therefore, small molecule inhibitors, capable of disrupting HR in protozoan parasites, represent potential therapeutic options. A number of small molecule inhibitors were identified that disrupt the activities of the human recombinase RAD51. Recent studies have examined the effect of two of these molecules on the Entamoeba recombinases. Here, we discuss the current understandings of HR in the protozoan parasites Trypanosoma, Leishmania, Plasmodium, and Entamoeba, and we review the small molecule inhibitors known to disrupt human RAD51 activity

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

This paper contains data related to the research article titled “Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase” (Kelso et al., in press) [1]. The known and putative amino acid sequence of Rad51, the central enzyme of homologous recombination, from nineteen different higher and lower eukaryotic organisms was analyzed. Here, we show amino acid conservation using a multiple sequence alignment, overall sequence identities using a percent identity matrix, and the evolutionary relationship between organisms using a neighbor-joining tree

Hed1 regulates Rad51-mediated recombination via a novel mechanism

Two RecA orthologs, Rad51 and Dmc1, mediate homologous recombination in meiotic cells. During budding yeast meiosis, Hed1 coordinates the actions of Rad51 and Dmc1 by down-regulating Rad51 activity. It is thought that Hed1-dependent attenuation of Rad51 facilitates formation of crossovers that are necessary for the correct segregation of chromosomes at the first meiotic division. We purified Hed1 in order to elucidate its mechanism of action. Hed1 binds Rad51 with high affinity and specificity. We show that Hed1 does not adversely affect assembly of the Rad51 presynaptic filament, but it specifically prohibits interaction of Rad51 with Rad54, a Swi2/Snf2-like factor that is indispensable for Rad51-mediated recombination. In congruence with the biochemical results, Hed1 prevents the recruitment of Rad54 to a site-specific DNA double-strand break in vivo but has no effect on the recruitment of Rad51. These findings shed light on the function of Hed1 and, importantly, unveil a novel mechanism for the regulation of homologous recombination