Rad51 Protein Research Articles

RAD51 Inhibition Shows Antitumor Activity in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), the major type of liver cancer, causes a high annual mortality worldwide. RAD51 is the critical recombinase responsible for homologous recombination (HR) repair in DNA damage. In this study, we identified that RAD51 was upregulated in HCC and that RAD51 silencing or inhibition reduced the proliferation, migration, and invasion of HCC cells and enhanced cell apoptosis and DNA damage. HCC cells with the combinatorial treatments of RAD51 siRNA or inhibitor and sorafenib demonstrated a synergistic effect in inhibiting HCC cell proliferation, migration, and invasion, as well as inducing cell apoptosis and DNA damage. Single RAD51 silencing or sorafenib reduced RAD51 protein expression and weakened HR efficiency, and their combination almost eliminated RAD51 protein expression and inhibited HR efficiency further. An in vivo tumor model confirmed the RAD51 inhibitor's antitumor activity and synergistic antitumor activity with sorafenib in HCC. RNA-Seq and gene set enrichment analysis (GSEA) in RAD51-inactivated Huh7 cells indicated that RAD51 knockdown upregulated cell apoptosis and G1/S DNA damage checkpoint pathways while downregulating mitotic spindle and homologous recombination pathways. Our findings suggest that RAD51 inhibition exhibits antitumor activities in HCC and synergizes with sorafenib. Targeting RAD51 may provide a novel therapeutic approach in HCC.

Racial differences in RAD51 expression are regulated by miRNA-214-5P and its inhibition synergizes with olaparib in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) affects young women and is the most aggressive subtype of breast cancer (BC). TNBCs disproportionally affect women of African-American (AA) descent compared to other ethnicities. We have identified DNA repair gene RAD51 as a poor prognosis marker in TNBC and its posttranscriptional regulation through microRNAs (miRNAs). This study aims to delineate the mechanisms leading to RAD51 upregulation and develop novel therapeutic combinations to effectively treat TNBCs and reduce disparity in clinical outcomes.MethodsAnalysis of TCGA data for BC cohorts using the UALCAN portal and PrognoScan identified the overexpression of RAD51 in TNBCs. miRNA sequencing identified significant downregulation of RAD51-targeting miRNAs miR-214-5P and miR-142-3P. RT-PCR assays were used to validate the levels of miRNAs and RAD51, and immunohistochemical and immunoblotting techniques were used similarly for RAD51 protein levels in TNBC tissues and cell lines. Luciferase assays were performed under the control of RAD51 3’-UTR to confirm that miR-214-5P regulates RAD51 expression. To examine the effect of miR-214-5P-mediated downregulation of RAD51 on homologous recombination (HR) in TNBC cells, Dr-GFP reporter assays were performed. To assess the levels of olaparib-induced DNA damage responses in miR-214-5P, transfected cells, immunoblots, and immunofluorescence assays were used. Furthermore, COMET assays were used to measure DNA lesions and colony assays were performed to assess the sensitivity of BRCA-proficient TNBC cells to olaparib.ResultsIn-silico analysis identified upregulation of RAD51 as a poor prognostic marker in TNBCs. miRNA-seq data showed significant downregulation of miR-214-5P and miR-142-3P in TNBC cell lines derived from AA women compared to Caucasian-American (CA) women. miR-214-5P mimics downregulated RAD51 expression and induces HR deficiency as measured by Dr-GFP assays in these cell lines. Based on these results, we designed a combination treatment of miR-214-5P and olaparib in HR-proficient AA TNBC cell lines using clonogenic survival assays. The combination of miR-214-5P and olaparib showed synergistic lethality compared to individual treatments in these cell lines.ConclusionsOur studies identified a novel epigenetic regulation of RAD51 in TNBCs by miR-214-5P suggesting a novel combination therapies involving miR-214-5P and olaparib to treat HR-proficient TNBCs and to reduce racial disparity in therapeutic outcomes.

Depletion of CNOT4 modulates the DNA damage responses following ionizing radiation (IR).

The Ccr4-Not complex (CNOT complex in mammals) is a unique and highly conserved complex with numerous cellular functions. Until now, there has been relatively little known about the importance of the CNOT complex subunits in the DNA damage response (DDR) in mammalian cells. CNOT4 is a subunit of the complex with E3 ubiquitin ligase activity that interacts transiently with the CNOT1 subunit. Here, we attempt to investigate the role of human CNOT4 subunit in the DDR in human cells. In this study, cell viability in the absence of CNOT4 was assessed using a Cell Titer-Glo Luminescence assay up to 4 days post siRNA transfection. In a further experiment, CNOT4-depleted HeLa cells were exposed to 3Gy ionizing radiation (IR). Ataxia telangiectasia-mutated (ATM) and ATM Rad3-related (ATR) signaling pathways were then investigated by western blotting for phosphorylated substrates. In addition, foci formation of histone 2A family member X (γH2AX), replication protein A (RPA), TP53 binding protein 1 (53BP1), and DNA repair protein RAD51 homolog 1 was also determined by immunofluorescence microscopy comparing control and CNOT4-depleted HeLa cells 0, 8, and 24 h post IR treatment. Our results from cell viability assays showed a significant reduction of cell growth activity at 24 (P value 0.02) and 48 h (P value 0.002) post siRNA. Western blot analysis showed slightly reduced or slightly delayed DDR signaling in CNOT4-depleted HeLa cells after IR. More significantly, we observed increased formation of γH2AX, RPA, 53BP1, and RAD51 foci after IR in CNOT4-depleted cells compared with the control cells. We conclude that depletion of CNOT4 affects various aspects of the cellular response to DNA damage.

ATM depletion induces proteasomal degradation of FANCD2 and sensitizes neuroblastoma cells to PARP inhibitors

BackgroundGenomic alterations, including loss of function in chromosome band 11q22-23, are frequently observed in neuroblastoma, which is the most common extracranial childhood tumour. In neuroblastoma, ATM, a DNA damage response-associated gene located on 11q22-23, has been linked to tumorigenicity. Genetic changes in ATM are heterozygous in most tumours. However, it is unclear how ATM is associated with tumorigenesis and cancer aggressiveness.MethodsTo elucidate its molecular mechanism of action, we established ATM-inactivated NGP and CHP-134 neuroblastoma cell lines using CRISPR/Cas9 genome editing. The knock out cells were rigorously characterized by analyzing proliferation, colony forming abilities and responses to PARP inhibitor (Olaparib). Western blot analyses were performed to detect different protein expression related to DNA repair pathway. ShRNA lentiviral vectors were used to knockdown ATM expression in SK-N-AS and SK-N-SH neuroblastoma cell lines. ATM knock out cells were stably transfected with FANCD2 expression plasmid to over-expressed the FANCD2. Moreover, knock out cells were treated with proteasome inhibitor MG132 to determine the protein stability of FANCD2. FANCD2, RAD51 and γH2AX protein expressions were determined by Immunofluorescence microscopy.ResultsHaploinsufficient ATM resulted in increased proliferation (p < 0.01) and cell survival following PARP inhibitor (olaparib) treatment. However, complete ATM knockout decreased proliferation (p < 0.01) and promoted cell susceptibility to olaparib (p < 0.01). Complete loss of ATM suppressed the expression of DNA repair-associated molecules FANCD2 and RAD51 and induced DNA damage in neuroblastoma cells. A marked downregulation of FANCD2 expression was also observed in shRNA-mediated ATM-knockdown neuroblastoma cells. Inhibitor experiments demonstrated that the degradation of FANCD2 was regulated at the protein level through the ubiquitin–proteasome pathway. Reintroduction of FANCD2 expression is sufficient to reverse decreased proliferation mediated by ATM depletion.ConclusionsOur study revealed the molecular mechanism underlying ATM heterozygosity in neuroblastomas and elucidated that ATM inactivation enhances the susceptibility of neuroblastoma cells to olaparib treatment. These findings might be useful in the treatment of high-risk NB patients showing ATM zygosity and aggressive cancer progression in future.

Abstract 6108: Novel functional dSTRIDE-HR assays to report on the status of homologous recombination repair in cancer cells

Abstract Since the early days of the synthetic lethality concept in DNA Damage Response the status of homologous recombination (HR) repair in cancer cells have been the focus of attention of researchers and clinicians. While different approaches exist, such as the RAD51 immunofluorescence (IF) or HRD genomic assays, functional biomarkers that can assess HR proficiency are missing. We report here the development, optimization and validation of two complementary, HR-specific functional assays. The assays, which are based on the STRIDE platform technology, detect double-strand DNA breaks localized in close proximity to RPA or RAD51 proteins. The optimization phase of assay development was performed in U2OS cells. First, repeatability (intra-run variation) and reproducibility (inter-run variation) of the assays were measured in untreated cells. Then, a series of technical negative controls was performed which have shown that the number of false-positive readouts is below 10% of the total number of signals. Finally, treatment of cells with compounds known to induce double-strand DNA breaks (etoposide and cisplatin) resulted in statistically significant increase in the number of detected dSTRIDE-RAD51 and dSTRIDE-RPA foci when compared to untreated controls. The assays were further validated in NCI-H661 (BRCA2 wild-type) and NCI-H169 (BRCA2 KO) cell line pair. The cells were treated with two concentrations of etoposide and the readouts from dSTRIDE, detecting the total pool of DSBs and dSTRIDE-HR assays were compared. In NCI-H661 cells, treatment with etoposide resulted in an increase in the number of double-strand breaks detected by dSTRIDE and as expected, more DSBs were formed after treatment with the higher concentration. dSTRIDE-HR assays confirmed that approximately 15% and 10% of these DSBs contain RPA and RAD51 proteins, respectively. In NCI-H169 cells etoposide produced a stronger reaction with even more DSBs detected by dSTRIDE, but importantly, no increase in the number of dSTRIDE-RAD51 foci was observed. dSTRIDE-RPA foci increased after treatment hinting that this step of HR remains unperturbed. Interestingly, the number of dSTRIDE-RAD51 foci in untreated cells was comparable between the two cell lines. In conclusion, we show here that two newly developed dSTRIDE-HR assays are well validated and can be successfully applied to report on the status of homologous recombination repair in different cell models. Citation Format: Kamil Solarczyk, Agnieszka Waligórska, Karolina Uznańska, Zsombor Prucsi, Olga Wójcikowska, Ewelina Matuszyk, Magdalena Bartyńska, Agata Kitlińska, Aleksandra Bober, Franek Sierpowski, Maja Białecka, Monika Jarosz, Malgorzata Szczygiel, Szymon Koman, Karolina Korpanty, Lukasz Beben, Lukasz Bandzarewicz, Przemyslaw Stachura, Magdalena Kordon-Kiszala. Novel functional dSTRIDE-HR assays to report on the status of homologous recombination repair in cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6108.

Abstract 310: CHAF1A promotes the translesion DNA synthesis pathway in response to DNA replication stress

Abstract Objective: Translesion DNA synthesis (TLS) pathway allows cancer cells to bypass the damage, allowing the blocked replication fork to move forward and helping cancer cells avoid genomic instability and cell death caused. Although some PCNA partners have previously been reported to participate in the TLS pathway, the mechanisms that regulate the TLS pathway remain unclear. Here, we revealed that CHAF1A is a new important regulator of TLS pathway. Methods: Cell models of CHAF1A gene knockdown (A549, KYSE510, KYSE450, etc.) were constructed by siRNA/shRNA knockdown method. We used DNA damage stimulation (HU, APH and UV) combined with western blotting to explore the effect of CHAF1A on PCNA monoubiquitination. The interaction of CHAF1A and its partners was demonstrated by immunoprecipitation and Duolink PLA assay. Cell fractionation assay was employed to measure protein binding to chromatin. DNA fiber assay was used to observe the condition of DNA replication fork. Micronucleus analysis was used to quantify cell micronucleus. The sensitivity of cells to DNA replication stress was tested by cloning formation assay and cell death assay. Kaplan-Meier survival analysis were used to analyze the clinical significance of CHAF1A expression. Results: CHAF1A enhances the interaction between PCNA and E3 ubiquitin protein ligase RAD18 and promotes PCNA monoubiquitination, thereby promoting the recruitment of Y-family DNA polymerase Pol η and enhancing cancer cell resistance to stimuli that trigger replication fork blockade. Mechanistically, CHAF1A-mediated PCNA monoubiquitination is independent of CHAF1A-PCNA interaction. CHAF1A interacts with both RAD18 and replication protein A2 (RPA2), mediating RAD18 binding on chromatin in response to DNA replication stress. Moreover, high expression of CHAF1A is significantly associated with poor prognosis in cancer patients. Conclusion: CHAF1A is a key TLS pathway regulator that is essential for cancer cell survival under DNA replication stress. The overall survival time of patients with high CHAF1A expression in lung, liver and esophageal cancers was significantly reduced. CHAF1A can be used as a key potential anti-tumor target, which has important clinical significance for enhancing the sensitivity of patients to radiotherapy and chemotherapy (This work was supported by the National Natural Science Foundation of China (82273108 and 82173034), the Innovative Team Grant of Guangdong Department of Education (2021KCXTD005), the Science and Technology Special Fund project of Guangdong Province in 2021 (No. 210729156901797) and the 2020 Li Ka Shing Foundation Cross-Disciplinary Research Grant (2020LKSFG07B)). Citation Format: Bing Wen, Li-Yan Xu, En-Min Li. CHAF1A promotes the translesion DNA synthesis pathway in response to DNA replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 310.

Abstract 2839: Methionine restriction radiosensitizes KRAS mutant rectal cancer

Abstract Purpose/Objective(s): Radiation therapy (RT) is the standard of care in patients with locally advanced rectal cancer, but not all patients respond equally to RT. KRAS-mutated (KRASmut) cancer cells are highly radioresistance and require higher levels of methionine, an essential amino acid, compared to KRAS wildtype (KRASwt) and normal cells, and diet restriction of methionine has been shown to potentiate the effects of radiation in vivo. We hypothesized that methionine restriction (MR) could radiosensitize KRASmut rectal cancer cells. Materials/Methods: HCT116 KRASG13D/+ and SW48 KRASwt colorectal carcinoma isogenic cell lines and their isogenic derivative cell lines HCT 116 KRAS+/- and SW48 KRASG13D were obtained from Horizon Discovery Ltd (Cambridge, UK). Radiation clonogenic assays, immunofluorescence, and western blot analysis of key DNA damage response proteins were performed in the KRASmut vs. KRASwt colorectal cells treated with ionizing radiation (IR, 2-6 Gy) at increasing time points following pre-treatment with methionine control (MC) (200 µM) or MR (40-fold reduction in methionine, 5 µM) media for 48 hours. One hundred thousand luciferase-expressing KRASwt or KRASmut SW48 cells were implanted orthotopically in the rectum of athymic nude mice. After confirmation of tumor formation at one-week post-injection, the mouse diet was changed to either an MC (0.84% methionine w/w) or MR (0.12% methionine w/w) diet for a total of two weeks. Rectal tumors were treated on an Xstrahl small animal radiation research platform (SARRP) to a dose of 25 Gy in 5 daily fractions (1 cm x 1 cm collimator, isocenter at anal verge) during the second week of diet modification. Tumor growth was measured via bioluminescence on IVIS Lumina every two weeks for four weeks post-radiation. Results: MR significantly increased radiosensitization compared to MC in both HCT116 and SW48 cells in vitro. KRASmut cells showed a 20-30% increase in radiation in the dose enhancement ratio compared to the KRASwt cells. The protein expression and nuclear foci per cell of the DNA damage marker γH2AX were significantly higher in the KRASmut cells compared to KRASwt cells following 5 Gy of RT at 6 hours. Protein expression of homologous recombination pathway proteins RAD51 and the CtBP interacting protein (CtIP) were higher at baseline in the KRASmut cells than KRASwt. MR substantially reduced the expression of these proteins following RT. Mice harboring SW48 KRASmut rectal tumors treated with 5 Gy x 5 RT and MR showed a significant increase in the complete response rate compared to MC (80% vs. 10%, p=0.02). In the mice harboring SW48 KRASwt rectal tumors, there was no significant difference in response rate between MR vs. MC (40% vs. 30%, p=NS). Conclusion: Our study indicates a novel strategy to radiosensitize resistant KRAS-mutated rectal cancer through MR. Studies testing the mechanism of KRAS-dependent methionine-mediated DNA repair is ongoing. Citation Format: Adam R. Wolfe, Sarita Garg, Stetson Van Matre, Robin Eluvathingal, Oscar Zuniga, Henrique Rodrigues, Nükhet Aykin-Burns, Isabelle Racine Miousse. Methionine restriction radiosensitizes KRAS mutant rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2839.

Novel Insights into RAD52's Structure, Function, and Druggability for Synthetic Lethality and Innovative Anticancer Therapies.

In recent years, the RAD52 protein has been highlighted as a mediator of many DNA repair mechanisms. While RAD52 was initially considered to be a non-essential auxiliary factor, its inhibition has more recently been demonstrated to be synthetically lethal in cancer cells bearing mutations and inactivation of specific intracellular pathways, such as homologous recombination. RAD52 is now recognized as a novel and critical pharmacological target. In this review, we comprehensively describe the available structural and functional information on RAD52. The review highlights the pathways in which RAD52 is involved and the approaches to RAD52 inhibition. We discuss the multifaceted role of this protein, which has a complex, dynamic, and functional 3D superstructural arrangement. This complexity reinforces the need to further investigate and characterize RAD52 to solve a challenging mechanistic puzzle and pave the way for a robust drug discovery campaign.

Importance of Germline and Somatic Alterations in Human MRE11, RAD50, and NBN Genes Coding for MRN Complex

The MRE11, RAD50, and NBN genes encode for the nuclear MRN protein complex, which senses the DNA double strand breaks and initiates the DNA repair. The MRN complex also participates in the activation of ATM kinase, which coordinates DNA repair with the p53-dependent cell cycle checkpoint arrest. Carriers of homozygous germline pathogenic variants in the MRN complex genes or compound heterozygotes develop phenotypically distinct rare autosomal recessive syndromes characterized by chromosomal instability and neurological symptoms. Heterozygous germline alterations in the MRN complex genes have been associated with a poorly-specified predisposition to various cancer types. Somatic alterations in the MRN complex genes may represent valuable predictive and prognostic biomarkers in cancer patients. MRN complex genes have been targeted in several next-generation sequencing panels for cancer and neurological disorders, but interpretation of the identified alterations is challenging due to the complexity of MRN complex function in the DNA damage response. In this review, we outline the structural characteristics of the MRE11, RAD50 and NBN proteins, the assembly and functions of the MRN complex from the perspective of clinical interpretation of germline and somatic alterations in the MRE11, RAD50 and NBN genes.

In Silico-Based Structural Evaluation to Categorize the Pathogenicity of Mutations Identified in the RAD Class of Proteins.

RAD genes, known as double-strand break repair proteins, play a major role in maintaining the genomic integrity of a cell by carrying out essential DNA repair functions via double-strand break repair pathways. Mutations in the RAD class of proteins show high susceptibility to breast and ovarian cancers; however, adequate research on the mutations identified in these genes has not been extensively reported for their deleterious effects. Changes in the folding pattern of RAD proteins play an important role in protein-protein interactions and also functions. Missense mutations identified from four cancer databases, cBioPortal, COSMIC, ClinVar, and gnomAD, cause aberrant conformations, which may lead to faulty DNA repair mechanisms. It is therefore necessary to evaluate the effects of pathogenic mutations of RAD proteins and their subsequent role in breast and ovarian cancers. In this study, we have used eight computational prediction servers to analyze pathogenic mutations and understand their effects on the protein structure and function. A total of 5122 missense mutations were identified from four different cancer databases, of which 1165 were predicted to be pathogenic using at least five pathogenicity prediction servers. These mutations were characterized as high-risk mutations based on their location in the conserved domains and subsequently subjected to structural stability characterization. The mutations included in the present study were selected from clinically relevant mutants in breast cancer pedigrees. Comparative folding patterns and intra-atomic interaction results showed alterations in the structural behavior of RAD proteins, specifically RAD51C triggered by mutations G125V and L138F and RAD51D triggered by mutations S207L and E233G.

The multi-BRCT domain protein DDRM2 promotes the recruitment of RAD51 to DNA damage sites to facilitate homologous recombination.

DNA double-strand breaks (DSBs) are the most toxic form of DNA damage in cells. Homologous recombination (HR) is an error-free repair mechanism for DSBs as well as a basis for gene targeting using genome-editing techniques. Despite the importance of HR, the HR mechanism in plants is poorly understood. Through genetic screens for DNA damage response mutants (DDRMs), we find that the Arabidopsis ddrm2 mutant is hypersensitive to DSB-inducing reagents. DDRM2 encodes a protein with four BRCA1 C-terminal (BRCT) domains and is highly conserved in plants including bryophytes, the earliest land plant lineage. The plant-specific transcription factor SOG1 binds to the promoter of DDRM2 and activates its expression. In consistence, the expression of DDRM2 is induced by DSBs in a SOG1-dependent manner. In support, genetic analysis suggests that DDRM2 functions downstream of SOG1. Similar to the sog1 mutant, the ddrm2 mutant shows dramatically reduced HR efficiency. Mechanistically, DDRM2 interacts with the core HR protein RAD51 and is required for the recruitment of RAD51 to DSB sites. Our study reveals that SOG1-DDRM2-RAD51 is a novel module for HR, providing a potential target for improving the efficiency of gene targeting.

Zn(II) to Ag(I) Swap in Rad50 Zinc Hook Domain Leads to Interprotein Complex Disruption through the Formation of Highly Stable Agx(Cys)y Cores.

The widespread application of silver nanoparticles in medicinal and daily life products increases the exposure to Ag(I) of thiol-rich biological environments, which help control the cellular metallome. A displacement of native metal cofactors from their cognate protein sites is a known phenomenon for carcinogenic and otherwise toxic metal ions. Here, we examined the interaction of Ag(I) with the peptide model of the interprotein zinc hook (Hk) domain of Rad50 protein from Pyrococcus furiosus, a key player in DNA double-strand break (DSB) repair. The binding of Ag(I) to 14 and 45 amino acid long peptide models of apo- and Zn(Hk)2 was experimentally investigated by UV-vis spectroscopy, circular dichroism, isothermal titration calorimetry, and mass spectrometry. The Ag(I) binding to the Hk domain was found to disrupt its structure via the replacement of the structural Zn(II) ion by multinuclear Agx(Cys)y complexes. The ITC analysis indicated that the formed Ag(I)-Hk species are at least 5 orders of magnitude stronger than the otherwise extremely stable native Zn(Hk)2 domain. These results show that Ag(I) ions may easily disrupt the interprotein zinc binding sites as an element of silver toxicity at the cellular level.

Goliath induces inflammation in obese mice by linking fatty acid β-oxidation to glycolysis.

Obesity is associated with metabolic disorders and chronic inflammation. However, the obesity-associated metabolic contribution to inflammatory induction remains elusive. Here, we show that, compared with lean mice, CD4+ T cells from obese mice exhibit elevated basal levels of fatty acid β-oxidation (FAO), which promote T cell glycolysis and thus hyperactivation, leading to enhanced induction of inflammation. Mechanistically, the FAO rate-limiting enzyme carnitine palmitoyltransferase 1a (Cpt1a) stabilizes the mitochondrial E3 ubiquitin ligase Goliath, which mediates deubiquitination of calcineurin and thus enhances activation of NF-AT signaling, thereby promoting glycolysis and hyperactivation of CD4+ T cells in obesity. We also report the specific GOLIATH inhibitor DC-Gonib32, which blocks this FAO-glycolysis metabolic axis in CD4+ T cells of obese mice and reduces the induction of inflammation. Overall, these findings establish a role of a Goliath-bridged FAO-glycolysis axis in mediating CD4+ T cell hyperactivation and thus inflammation in obese mice.

Issue Information

FEBS Open BioVolume 13, Issue 3 p. 393-395 Issue InformationOpen Access Issue Information First published: 07 March 2023 https://doi.org/10.1002/2211-5463.13426AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Cover Illustration The full-length human RAD52 protein forms an undecameric ring structure as revealed by cryo-electron microscopy. See the article by Kinoshita et al. pp. 408-418. Volume13, Issue3March 2023Pages 393-395 RelatedInformation

Divalent metal cofactors differentially modulate RadA-mediated strand invasion and exchange in Saccharolobus solfataricus.

Central to the universal process of recombination, RecA family proteins form nucleoprotein filaments to catalyze production of heteroduplex DNA between substrate ssDNAs and template dsDNAs. ATP binding assists the filament in assuming the necessary conformation for forming heteroduplex DNA, but hydrolysis is not required. ATP hydrolysis has two identified roles which are not universally conserved: promotion of filament dissociation and enhancing flexibility of the filament. In this work, we examine ATP utilization of the RecA family recombinase SsoRadA from Saccharolobus solfataricus to determine its function in recombinase-mediated heteroduplex DNA formation. Wild-type SsoRadA protein and two ATPase mutant proteins were evaluated for the effects of three divalent metal cofactors. We found that unlike other archaeal RadA proteins, SsoRadA-mediated strand exchange is not enhanced by Ca2+. Instead, the S. solfataricus recombinase can utilize Mn2+ to stimulate strand invasion and reduce ADP-binding stability. Additionally, reduction of SsoRadA ATPase activity by Walker Box mutation or cofactor alteration resulted in a loss of large, complete strand exchange products. Depletion of ADP was found to improve initial strand invasion but also led to a similar loss of large strand exchange events. Our results indicate that overall, SsoRadA is distinct in its use of divalent cofactors but its activity with Mn2+ shows similarity to human RAD51 protein with Ca2+.

Biochemical characterization of the RNA-binding and RNA-DNA strand exchange activities of the human RAD52 protein.

RAD52 is a single-stranded DNA (ssDNA) binding protein that functions in the repair of DNA double-strand breaks (DSBs) by promoting the annealing of complementary DNA strands. RAD52 may also play an important role in an RNA transcript-dependent type of DSB repair, in which it reportedly binds to RNA and mediates the RNA-DNA strand exchange reaction. However, the mechanistic details of these functions are still unclear. In the present study, we utilized the domain fragments of RAD52 to biochemically characterize the single-stranded RNA (ssRNA) binding and RNA-DNA strand exchange activities of RAD52. We found that the N-terminal half of RAD52 is primarily responsible for both activities. By contrast, significant differences were observed for the roles of the C-terminal half in RNA-DNA and DNA-DNA strand exchange reactions. The C-terminal fragment stimulated the inverse RNA-DNA strand exchange activity displayed by the N-terminal fragment in trans, whereas the trans stimulatory effect by the C-terminal fragment was not observed in the inverse DNA-DNA or forward RNA-DNA strand exchange reactions. These results suggest the specific function of the C-terminal half of RAD52 in RNA-templated DSB repair.

Anti-tumour and radiosensitising effects of PARP inhibitor on cervical cancer xenografts

This study evaluated the radiosensitising effect of niraparib; a poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor on HeLa cervical cancer cells in nude mice and explored its possible mechanism. Twenty-four 3–5-week-old female BALB/c nude mice, inoculated with HeLa cells into the right hind leg, were randomly assigned into eight groups with three mice per group and treated. The tumour volume was significantly reduced under niraparib + radiotherapy combination as compared to monotherapy and untreated mice. The tumour growth was significantly delayed by 23.33–39 days when treated with combination therapy (p<.05). Further, univariate analysis revealed prolonged time for tumour growth when radiotherapy was followed by niraparib (I.G.) rather than niraparib (I.P.) (p=.003). Combination therapy reduced levels of PARP-1 precursor, PARP-1 splicer, PAR and RAD51 protein with high expression of γ-H2AX/CC3 and low expression of Ki-67. Niraparib in combination with radiotherapy can enhance the formation of DNA double strand breaks in HeLa cells and up regulate the expression of γ-H2AX/CC3. IMPACT STATEMENT What is already known on this subject? Asia has the highest incidence of cervical cancer (58.2%). Poly(adenosine diphosphate-ribose) polymerases (PARPs) are family of enzymes involved in single-strand break (SSB) and double-strand break (DSB) repair pathways. Niraparib is an effective inhibitor of both PARP-1 and PARP-2 and has the ability to cross the blood–brain barrier. What the results of this study add? Our study demonstrated that the combination of niraparib and radiotherapy can significantly enhance the cytotoxicity induced by radiotherapy. The inhibition effect of radiotherapy combined with niraparib on the tumour growth of mice was prominent, thereby establishing the radio-sensitisation activity of niraparib. What are the implications of these findings for clinical practice and/or further research? Niraparib can improve the cytotoxic effect of radiotherapy by increasing the formation of DSBs and up regulating the expression of apoptotic protein in HeLa cells.

The cryo-EM structure of full-length RAD52 protein contains an undecameric ring.

The human RAD52 protein, which forms an oligomeric ring structure, is involved in DNA double-strand break repair. The N-terminal half of RAD52 is primarily responsible for self-oligomerisation and DNA binding. Crystallographic studies have revealed the detailed structure of the N-terminal half. However, only low-resolution structures have been reported for the full-length protein, and thus the structural role of the C-terminal half in self-oligomerisation has remained elusive. In this study, we determined the solution structure of the human RAD52 protein by cryo-electron microscopy (cryo-EM), at an average resolution of 3.5 Å. The structure revealed an undecameric ring that is nearly identical to the crystal structures of the N-terminal half. The cryo-EM map for the C-terminal half was poorly defined, indicating that the region is intrinsically disordered. The present cryo-EM structure provides important insights into the mechanistic roles played by the N-terminal and C-terminal halves of RAD52 during DNA double-strand break repair.

The molecular mechanism for TERRA recruitment and annealing to telomere.

Telomeres are DNA-protein complexes that protect the ends of linear chromosomes. Telomeres are composed of tandem repeat sequences of duplexed (TTAGGG)n, ending with a 3’ single stranded (TTAGGG)n sequence bound to telomere specific proteins known as shelterin complex. In healthy somatic cells, progressive loss of telomeres results in cellular senescence. In 85% of cancer cells, however telomeres are elongated by telomerase, while the remaining 15% of cancers use alternative lengthening of telomeres (ALT). In ALT (+) cancers, Telomeric repeat containing RNA (TERRA), a noncoding RNA that is transcribed from telomeres is overexpressed. TERRA trans anneals by invading the telomeric duplex to form an R-loop. Recent study showed that R-loop formation is promoted by Rad51 and antagonized by RNaseH1. Despite the evidence of TERRA's role in telomere structure and function, our molecular understanding of TERRA is limited. Here, we elucidate the molecular mechanism underlying TERRA recruitment and invasion into telomeres in the context of shelterin, RAD51 and RNaseH1 using single molecule (sm) assays. Using smFRET we show that TERRA trans anneals to telomeric DNA and exhibits dynamic movement that is stabilized by TRF2. Additionally, TERRA annealing to the telomeric duplex forms a triplex structure. Sm-colocalization assays show that the presence of non-TERRA sequence, which represents the sub-telomeric DNA and the presence of G-quadruplex telomeric overhang enhances TERRA binding to telomeric duplex. We also studied the mechanism underlying TERRA recruitment and invasion by Rad51 and shelterin proteins. Our result shows that RAD51-TERRA complex invades telomere duplex more efficiently than TERRA alone. Additionally, TRF2 increases TERRA affinity to telomeric duplex and protects it from RNaseH1 digestion. In contrast, TRF1 represses TERRA binding to telomeric duplex and does not provide protection against RNaseH1. We demonstrate the molecular mechanism underpinning TERRA recruitment and annealing to telomere.

Microtubule-associated proteins MAP7 and MAP7D1 promote DNA double-strand break repair in the G1 cell cycle phase

The DNA-damage response is a complex signaling network that guards genomic integrity. The microtubule cytoskeleton is involved in the repair of DNA double-strand breaks; however, little is known about which cytoskeleton-related proteins are involved in DNA repair and how. Using quantitative proteomics, we discovered that microtubule associated proteins MAP7 and MAP7D1 interact with several DNA repair proteins including DNA double-strand break repair proteins RAD50, BRCA1 and 53BP1. We observed that downregulation of MAP7 and MAP7D1 leads to increased phosphorylation of p53 after γ-irradiation. Moreover, we determined that the downregulation of MAP7D1 leads to a strong G1 arrest and that the downregulation of MAP7 and MAP7D1 in G1 arrested cells negatively affects DNA repair, recruitment of RAD50 to chromatin and localization of 53BP1 to the sites of damage. These findings describe for the first time a novel function of MAP7 and MAP7D1 in cell cycle regulation and repair of DNA double-strand breaks.