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.
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.
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.
Parkinson's disease (PD) is the most common neurodegenerative movement disorder and neuroprotective interventions remain elusive. High throughput biomarkers aimed to stratify patients based on shared etiology is one critical path to the success of disease-modifying therapies in clinical trials. Mitochondrial dysfunction plays a prominent role in the pathogenesis of PD. Previously, we found brain region-specific mitochondrial DNA (mtDNA) damage accumulation in neuronal and in vivo PD models, as well as human PD postmortem brain tissue. In this study, to investigate mtDNA damage as a potential blood biomarker for PD, we describe a novel Mito DNADX assay that allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, compatible with assessing large cohorts of patient samples. We found that levels of mtDNA damage were increased in blood derived from early-stage idiopathic PD patients or those harboring the pathogenic LRRK2 G2019S mutation compared to age-matched healthy controls. Given that increased mtDNA damage was also found in non-manifesting LRRK2 mutation carriers, mtDNA damage may begin to accumulate prior to a clinical PD diagnosis. LRRK2 kinase inhibition mitigated mtDNA damage in idiopathic PD models and patient-derived cells. The latter observations further substantiate a mechanistic role for wild-type LRRK2 kinase activity in idiopathic PD and support mtDNA damage reversal as a suitable approach to slow PD-related pathology. In light of recent advances in the field of precision medicine, the analysis of mtDNA damage as a blood-based patient stratification biomarker should be included in future clinical trials.
Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting over one million people in the US. Mutations in leucine‐rich repeat kinase 2 (LRRK2) are the most common cause of inherited and idiopathic PD. We were the first to show that mitochondrial DNA (mtDNA) damage is caused by the most common mutation in LRRK2 (G2019S) and inhibition of LRRK2 kinase activity restores mtDNA integrity in PD models. However, whether aberrant LRRK2 kinase activity due to PD‐linked mutations has broad impact on nuclear genome integrity is unknown. Using LRRK2G2019S/G2019S knock‐in (KI) human embryonic kidney 293 (HEK293) cells obtained by CRISPR/Cas9 gene editing, our preliminary results indicate nuclear DNA damage is increased, including DNA double‐strand breaks (DSBs) as assayed by a neutral comet assay. Consistent with DSB accumulation, we observed significantly increased γ‐H2AX and 53BP1 foci. ATM is activated by DSBs and phosphorylates several key proteins that initiate the DNA damage response, cell cycle arrest, DNA repair or apoptosis. We found that basal levels of ATM pS1981 are increased in this in vitro LRRK2 G2019S model, which is an autophosphorylation site that correlates with DNA damage‐mediated activation. Additionally, downstream substrates CHK2 (pT68), and P53 (pS15) are similarly increased in LRRK2G2019S/G2019S KI cells compared to isogenic wild‐type control cells, substantiating that the ATM‐mediated DNA damage response pathway has been up‐regulated with the LRRK2 G2019S mutation. Blocking either LRRK2 or ATM kinase activity pharmacologically, significantly reversed LRRK2 G2019S‐induced γ‐H2AX foci. Overall these results suggest DSBs accumulate in LRRK2 PD, which in turn activate a sustained ATM mediated DNA damage response, which may lead to cell cycle arrest, aberrant DNA repair, and/or cell death. Further understanding of the functional relationship between LRRK2 and ATM offers new molecular insights into PD pathogenesis.
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