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

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Novel attributes of Hed1 affect dynamics and activity of the Rad51 presynaptic filament during meiotic recombination

During meiosis, recombination events that occur between homologous chromosomes help prepare the chromosome pairs for proper disjunction in meiosis I. The concurrent action of the Rad51 and Dmc1 recombinases is necessary for an interhomolog bias. Notably, the activity of Rad51 is tightly controlled, so as to minimize the use of the sister chromatid as recombination partner. We demonstrated recently that Hed1, a meiosis-specific protein in Saccharomyces cerevisiae, restricts the access of the recombinase accessory factor Rad54 to presynaptic filaments of Rad51. We now show that Hed1 undergoes self-association in a Rad51-dependent manner and binds ssDNA. We also find a strong stabilizing effect of Hed1 on the Rad51 presynaptic filament. Biochemical and genetic analyses of mutants indicate that these Hed1 attributes are germane for its recombination regulatory and Rad51 presynaptic filament stabilization functions. Our results shed light on the mechanism of action of Hed1 in meiotic recombination control

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 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

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 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