Contributions of the Human SSB Complex and MEI5-SWI5 Complex to Homologous Recombination

DNA double-strand breaks (DSBs) are the most threatening type of DNA damage in a cell. Homologous recombination (HR) is the most accurate repair mechanism for DSBs, and if HR fails, the integrity of the genome can be compromised. Two recombinases, RAD51 and DMC1, are vital for HR but require assistance for HR to proceed efficiently and accurately. Several proteins, including mediators, single-strand binding proteins, and accessory proteins, have been shown to function in the HR with the recombinases. Mediators are responsible for overcoming inhibition caused by the single-strand binding protein, Replication protein A (RPA). Accessory proteins assist the recombinases through DSB localization, ATP hydrolysis, filament stabilization and several other functions. In addition to RPA, higher eukaryotes possess two other SSBs, SSB1 and SSB2. Both hSSBs maintain genomic integrity through participation in the HR pathway. It was previously demonstrated that hSSB1 stimulates RAD51 during D-loop formation. Additionally, the hSSBs maintain genomic integrity through the repair of stalled replication forks. In this dissertation, we present in Chapter 2 surprising activities of the hSSBs that support the recent genetic data implicating hSSB1 and hSSB2 in the repair of stalled replication forks. We demonstrated a functional interaction with the human polymerase η in D-loop extension and second-end capture. This is the first report of the hSSBs interaction with a polymerase and identifies a new function of the hSSBs in DNA double-strand break repair. We also report that hSSB1 and hSSB2 can anneal single-strand DNA and melt double-strand DNA. In Chapter 3, we examined the effect of CaCl2 and MgCl2 on hSSB D-loop formation and demonstrate that hSSB1 and hSSB2 can in fact form D-loops in the absence of the recombinase, RAD51. Both hSSB1 and hSSB2 form a heterotrimeric complex with Integrator subunit 3 (INTS3) and the Single-strand interacting protein 1 (hSSBIP1). We have purified the components and confirmed complex formation. The effect of the complex proteins on D-loop extension by hPol η will be interesting to examine in the future. The hMEI5-SWI5 ortholog in Saccharomyces cerevisiae functions as a mediator to scDMC1. To date, there have been no reports regarding hMEI5-SWI5 functionality with hDMC1. In Chapter 3, we examined the DNA binding activity of hMEI5 and hSWI5 individually and as a complex (Mei5-Swi5), in addition to demonstrating physical interaction with both DMC1 and RPA. Importantly, we report that hMEI5 but not hSWI5 retains mediator activity to hDMC1 using an in vitro homologous DNA pairing assay. This is the first biochemical report on hMEI5-hSWI5

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

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

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

<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

<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