Defining the role of Tip60 in the DNA damage response of glioma cell lines.

PURPOSE: Glioblastomas are resistant to conventional therapies, including radiotherapy. Our previous study proved that epigenetic regulation influences the radiation response of glioma cells. This study evaluated the role of the acetyltransferase Tip60 on the radiation response. MATERIAL AND METHODS: Tip60 expression was down-regulated by transfecting specific siRNA's in A7 and MO59K cells with high and low expression of Tip60, respectively, and its effect on survival was assessed. DNA repair was analyzed by foci scoring (γH2AX, Rad51, 53BP1, pATM). The interaction of Tip60 with ATM and DNA-PK was investigated using the specific inhibitors KU55933 and NU7441, respectively. RESULTS: Knockdown of Tip60 significantly (p < .001) reduced survival in both cell lines, but the effect was more pronounced in A7 cells. ATMi and DNA-PKi significantly reduced the surviving fraction following irradiation. However, no further effect of siTip60 on the radiosensitivity of ATMi treated A7 cells was observed. In contrast, DNA-PKi effectively enhanced the sensitizing effect of siTip60. Mechanistically, siTip60 reduced the number of initial Rad51 and ATM foci formation after irradiation and prevented their dissolution at 24 h. siTip60 had no impact on the formation of 53BP1 and γH2AX foci and did not further affect these end-points if combined with ATMi or DNA-PKi. CONCLUSIONS: Downregulation of Tip60 enhances the radiation sensitivity of both glioma cells and markedly elevates the radiation sensitivity when combined with DNA-PKi. Therefore, treatment with DNA-PK inhibitors represents a promising approach to augment the radiation sensitivity of glioma cell lines with deficient Tip60 activity in a synergistic manner.

RAD51 recombinase and its paralogs: orchestrating homologous recombination and unforeseen functions in protozoan parasites.

The DNA of protozoan parasites is highly susceptible to damage, either induced by environmental agents or spontaneously generated during cellular metabolism through reactive oxygen species (ROS). Certain phases of the cell cycle, such as meiotic recombination, and external factors like ionizing radiation (IR), ultraviolet light (UV), or chemical genotoxic agents further increase this susceptibility. Among the various types of DNA damage, double-stranded breaks (DSBs) are the most critical, as they are challenging to repair and can result in genetic instability or cell death. DSBs caused by environmental stressors are primarily repaired via one of two major pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). In multicellular eukaryotes, NHEJ predominates, but in unicellular eukaryotes such as protozoan parasites, HR seems to be the principal mechanism for DSB repair. The HR pathway is orchestrated by proteins from the RAD52 epistasis group, including RAD51, RAD52, RAD54, RAD55, and the MRN complex. This review focuses on elucidating the diverse roles and significance of RAD51 recombinase and its paralogs in protozoan parasites, such as Acanthamoeba castellanii, Entamoeba histolytica (Amoebozoa), apicomplexan parasites (Chromalveolata), Naegleria fowleri, Giardia spp., Trichomonas vaginalis, and trypanosomatids (Excavata), where they primarily function in HR. Additionally, we analyze the diversity of proteins involved in HR, both upstream and downstream of RAD51, and discuss the implications of these processes in parasitic protozoa.

Next-generation cell-penetrating antibodies for tumor targeting and RAD51 inhibition.

Monoclonal antibody therapies for cancer have demonstrated extraordinary clinical success in recent years. However, these strategies are thus far mostly limited to specific cell surface antigens, even though many disease targets are found intracellularly. Here we report studies on the humanization of a full-length, nucleic acid binding, monoclonal lupus-derived autoantibody, 3E10, which exhibits a novel mechanism of cell penetration and tumor specific targeting. Comparing humanized variants of 3E10, we demonstrate that cell uptake depends on the nucleoside transporter ENT2, and that faster cell uptake and superior in vivo tumor targeting are associated with higher affinity nucleic acid binding. We show that one human variant retains the ability of the parental 3E10 to bind RAD51, serving as a synthetically lethal inhibitor of homology-directed repair in vitro. These results provide the basis for the rational design of a novel antibody platform for therapeutic tumor targeting with high specificity following systemic administration.

FL118 Enhances Therapeutic Efficacy in Colorectal Cancer by Inhibiting the Homologous Recombination Repair Pathway through Survivin-RAD51 Downregulation.

Background/Objectives: Irinotecan, a camptothecin (CPT) derivative, is commonly used as a first-line therapy for colorectal cancer (CRC), but resistance remains a significant challenge. This study aims to explore the therapeutic potential of FL118, another CPT derivative, with a focus on overcoming resistance to irinotecan. Methods: The effects of FL118 on CRC cells were evaluated, and bioinformatics analysis was performed on RNA-seq data. Transfection was conducted to observe the knockdown effect of survivin, and the in vivo efficacy of FL118 was assessed using a xenograft model. Results: FL118 induces apoptosis, G2/M arrest, and DNA damage. A notable mechanism of action of FL118 is a reduction in survivin levels, which downregulates the expression of RAD51, a key marker of homologous recombination, and attenuates DNA repair processes. Given that SN38 is the active metabolite of irinotecan, FL118 reduces cell viability and RAD51 in SN38-resistant LOVO cells. Conclusions: Our findings provide effective insights into the antitumor activity of FL118 and its potential as a therapeutic agent for overcoming irinotecan resistance in CRC.

FIGL1 attenuates meiotic interhomolog repair and is counteracted by the RAD51 paralog XRCC2 and the chromosome axis protein ASY1 during meiosis.

Two recombinases, RAD51 and DMC1, catalyze meiotic break repair to ensure crossovers (COs) between homologous chromosomes (interhomolog) rather than between sisters (intersister). FIDGETIN-LIKE-1 (FIGL1) downregulates both recombinases. However, the understanding of how FIGL1 functions in meiotic repair remains limited. Here, we discover new genetic interactions of Arabidopsis thaliana FIGL1 that are important in vivo determinants of meiotic repair outcome. In figl1 mutants, compromising RAD51-dependent repair, either through the loss of RAD51 paralogs (RAD51B or XRCC2) or RAD54 or by inhibiting RAD51 catalytic activity, results in either unrepaired breaks or meiotic CO defects. Further, XRCC2 physically interacts with FIGL1 and partially counteracts FIGL1 activity for RAD51 focus formation. Our data indicate that RAD51-mediated repair mechanisms compensate FIGL1 dysfunction. FIGL1 is not necessary for intersister repair in dmc1 but is essential for the completion of meiotic repair in mutants such as asy1 that have impaired DMC1 functions and interhomolog bias. We show that FIGL1 attenuates interhomolog repair, and ASY1 counteracts FIGL1 to promote interhomolog recombination. Altogether, this study underlines that multiple factors can counteract FIGL1 activity to promote accurate meiotic repair.

Knocking down RAD51AP1 enhances chemosensitivity by inhibiting the self-renewal of CD133 positive ovarian cancer stem-like cells.

PURPOSE: This study was designed to investigate the function of RAD51AP1 in the self-renewal and chemosensitivity of CD133 positive (CD133+) ovarian cancer (OC) stem-like cells. METHODS: CD133+ (CD133 positive) OVCAR4 and CD133 negative (CD133-) OVCAR4 cells were separated from OVCAR4 by flow cytometry. Then, the separated CD133+OVCAR4 cells were divided into the following groups: Vector group; RAD51AP1 group; siNC group; si-RAD51AP1 group. Next, sphere-formation assay and colony forming assay were used to evaluate the self-renewal and proliferation ability of cells; western blot to detect the expression of RAD51AP1, transforming growth factor beta 1 (TGF-ß1) and SMAD4 proteins in tissues and cells; qRT-PCR to assess the mRNA levels of sex-determining region Y-box 2 (SOX2), octamer-binding transcription factor 4 (OCT4), NANOG and Kruppel-like factor 4 (KLF4). RESULTS: The performance of CD133+OVCAR4 cells was much better than that of CD133-OVCAR4 cells in sphere-formation assay and colony forming assay. Besides, compared with adjacent group and CD133-OVCAR4 cells, the expression level of RAD51AP1 increased significantly in OC group and CD133+OVCAR4 cells. Moreover, the over-expression of RAD51AP1 promoted the self-renewal and proliferation of CD133+OVCAR4 cells. On the contrary, knocking down the expression level of RAD51AP1 could inhibit the self-renewal and proliferation of CD133+OVCAR4 cells and improve the sensitivity of cells to chemotherapy drugs. CONCLUSION: The findings of this study showed that RAD51AP1 was highly expressed in OC tissue and CD133+OVCAR4 cells, and regulated the self-renewal and chemosensitivity of tumor cells through the TGF-ß1/SMAD4 signaling pathway.

High-resolution functional mapping of RAD51C by saturation genome editing.

Pathogenic variants in RAD51C confer an elevated risk of breast and ovarian cancer, while individuals homozygous for specific RAD51C alleles may develop Fanconi anemia. Using saturation genome editing (SGE), we functionally assess 9,188 unique variants, including >99.5% of all possible coding sequence single-nucleotide alterations. By computing changes in variant abundance and Gaussian mixture modeling (GMM), we functionally classify 3,094 variants to be disruptive and use clinical truth sets to reveal an accuracy/concordance of variant classification >99.9%. Cell fitness was the primary assay readout allowing us to observe a phenomenon where specific missense variants exhibit distinct depletion kinetics potentially suggesting that they represent hypomorphic alleles. We further explored our exhaustive functional map, revealing critical residues on the RAD51C structure and resolving variants found in cancer-segregating kindred. Furthermore, through interrogation of UK Biobank and a large multi-center ovarian cancer cohort, we find significant associations between SGE-depleted variants and cancer diagnoses.

Germline RAD51C and RAD51D Mutations in High-Risk Chinese Breast and/or Ovarian Cancer Patients and Families.

BACKGROUND: RAD51C and RAD51D are crucial in homologous recombination (HR) DNA repair. The prevalence of the RAD51C and RAD51D mutations in breast cancer varies across ethnic groups. Associations of RAD51C and RAD51D germline pathogenic variants (GPVs) with breast and ovarian cancer predisposition have been recently reported and are of interest. METHODS: We performed multi-gene panel sequencing to study the prevalence of RAD51C and RAD51D germline mutations among 3728 patients with hereditary breast and/or ovarian cancer (HBOC). RESULTS: We identified 18 pathogenic RAD51C and RAD51D mutation carriers, with a mutation frequency of 0.13% (5/3728) and 0.35% (13/3728), respectively. The most common recurrent mutation was RAD51D c.270_271dupTA; p.(Lys91Ilefs*13), with a mutation frequency of 0.30% (11/3728), which was also commonly identified in Asians. Only four out of six cases (66.7%) of this common mutation tested positive for homologous recombination deficiency (HRD). CONCLUSIONS: Taking the family studies in our registry and tumor molecular pathology together, we concluded that this relatively common RAD51D variant showed incomplete penetrance in our local Chinese community. Personalized genetic counseling emphasizing family history for families with this variant, as suggested at the UK Cancer Genetics Group (UKCGG) Consensus meeting, would also be appropriate in Chinese families.

Contribution of Radiation Sensitive Protein 51 Genotypes to Pterygium Risk in a Taiwanese Population.

BACKGROUND/AIM: In current literature, there is a notable lack of studies investigating the role of radiation-sensitive protein 51 (RAD-51) in pterygium diagnosis. Nevertheless, reports indicate elevated expression levels of RAD-51 among recurrent pterygium cases compared to those with primary pterygium. However, the genomic involvement of RAD-51 has yet to be explored in any population. This study aimed to assess the contribution of RAD-51 genotypes to pterygium risk in a representative Taiwanese population. MATERIALS AND METHODS: RAD-51 rs1801320 genotyping was successfully conducted in a Taiwanese cohort comprising 140 pterygium cases and 280 non-pterygium controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. RESULTS: The distribution of RAD-51 rs1801320 genotypes (GG, CG, and CC) in the pterygium group (70.0%, 25.7%, and 4.3%, respectively) did not significantly differ from that in the non-pterygium group (73.6%, 23.6%, and 2.8% for GG, CG, and CC genotypes, respectively; p for trend=0.6337). Carriers of the variant CG and CC RAD-51 rs1801320 genotypes exhibited 1.15- and 1.58-fold increased pterygium risk, respectively (95%CI=0.72-1.84 and 0.53-4.67, p=0.6552 and p=0.5914, respectively). In the dominant model, there appeared to be a slight association between variant genotypes CG and CC and pterygium risk (OR=1.19, 95%CI=0.76-1.87, p=0.0223). Allelic analysis revealed that the RAD-51 rs1801320 variant C allele was not significantly linked to pterygium risk (17.1% versus 14.6%, OR=1.20, 95%CI=0.82-1.78, p=0.3991). CONCLUSION: Variant genotypes at RAD-51 rs1801320 were firstly identified to associate with susceptibility to pterygium among Taiwanese individuals. Nonetheless, these findings warrant validation in larger and more diverse populations.

Screening of gastric cancer diagnostic biomarkers in the homologous recombination signaling pathway and assessment of their clinical and radiomic correlations.

BACKGROUND: Homologous recombination plays a vital role in the occurrence and drug resistance of gastric cancer. This study aimed to screen new gastric cancer diagnostic biomarkers in the homologous recombination pathway and then used radiomic features to construct a prediction model of biomarker expression to guide the selection of chemotherapy regimens. METHODS: Gastric cancer transcriptome data were downloaded from The Cancer Genome Atlas database. Machine learning methods were used to screen for diagnostic biomarkers of gastric cancer and validate them experimentally. Computed Tomography image data of gastric cancer patients and corresponding clinical data were downloaded from The Cancer Imaging Archive and our imaging centre, and then the Computed Tomography images were subjected to feature extraction, and biomarker expression prediction models were constructed to analyze the correlation between the biomarker radiomics scores and clinicopathological features. RESULTS: We screened RAD51D and XRCC2 in the homologous recombination pathway as biomarkers for gastric cancer diagnosis by machine learning, and the expression of RAD51D and XRCC2 was significantly positively correlated with pathological T stage, N stage, and TNM stage. Homologous recombination pathway blockade inhibits gastric cancer cell proliferation, promotes apoptosis, and reduces the sensitivity of gastric cancer cells to chemotherapeutic drugs. Our predictive RAD51D and XRCC2 expression models were constructed using radiomics features, and all the models had high accuracy. In the external validation cohort, the predictive models still had decent accuracy. Moreover, the radiomics scores of RAD51D and XRCC2 were also significantly positively correlated with the pathologic T, N, and TNM stages. CONCLUSIONS: The gastric cancer diagnostic biomarkers RAD51D and XRCC2 that we screened can, to a certain extent, reflect the expression status of genes through radiomic characteristics, which is of certain significance in guiding the selection of chemotherapy regimens for gastric cancer patients.

A glimpse into the hidden world of the flexible C-terminal protein binding domains of human RAD52.

Human RAD52 protein binds DNA and is involved in genomic stability maintenance and several forms of DNA repair, including homologous recombination and single-strand annealing. Despite its importance, there are very few structural details about the variability of the RAD52 ring size and the RAD52 C-terminal protein-protein interaction domains. Even recent attempts to employ cryogenic electron microscopy (cryoEM) methods on full-length yeast and human RAD52 do not reveal interpretable structures for the C-terminal half that contains the replication protein A (RPA) and RAD51 binding domains. In this study, we employed the monodisperse purification of two RAD52 deletion constructs and small angle X-ray scattering (SAXS) to construct a structural model that includes RAD52's RPA binding domain. This model is of interest to DNA repair specialists as well as for drug development against HR-deficient cancers.

Downregulation of Rad51 Expression and Activity Potentiates the Cytotoxic Effect of Osimertinib in Human Non-Small Cell Lung Cancer Cells.

INTRODUCTION: Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has shown significant clinical benefits in patients with EGFR-sensitizing mutations or the EGFR T790M mutation. The homologous recombination (HR) pathway is crucial for repairing DNA double-strand breaks (DSBs). Rad51 plays a central role in HR, facilitating the search for homology and promoting DNA strand exchange between homologous DNA molecules. Rad51 is overexpressed in numerous types of cancer cells. B02, a specific small molecule inhibitor of Rad51, inhibits the DNA strand exchange activity of Rad51. Previous studies have indicated that B02 disrupted Rad51 foci formation in response to DNA damage and inhibited DSBs repair in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo. However, the potential therapeutic effects of combining osimertinib with a Rad51 inhibitor are not well understood. The aim of this study was to elucidate whether the downregulation of Rad51 expression and activity can enhance the osimertinib-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells. METHODS: We used the MTS, trypan blue dye exclusion and colony-formation ability assay to determine whether osimertinib alone or in combination with B02 had cytotoxic effects on NSCLC cell lines. Real-time polymerase chain reaction was conducted to measure the amounts of Rad51 mRNA. The protein levels of phosphorylated AKT and Rad51 were determined by Western blot analysis. RESULTS: We found that osimertinib reduced Rad51 expression by inactivating AKT activity. Rad51 knockdown using small interfering RNA or AKT inactivation through the phosphatidylinositol 3-kinase inhibitor LY294002 or si-AKT RNA transfection enhanced the cytotoxic and growth inhibitory effects of osimertinib. In contrast, AKT-CA (a constitutively active form of AKT) vector-enforced expression could mitigate the cytotoxic and cell growth inhibitory effects of osimertinib. Furthermore, B02 significantly enhanced the cytotoxic and cell growth inhibitory effects of osimertinib in NSCLC cells. Compared to parental cells, the activation of AKT and Rad51 expression in osimertinib-resistant cells could not be significantly inhibited by osimertinib treatment. Moreover, the increased expression of Rad51 is associated with the resistance mechanism in osimertinib-resistant H1975 and A549 cells. CONCLUSION: Collectively, the downregulation of Rad51 expression and activity enhances the cytotoxic effect of osimertinib in human NSCLC cells.

Mechanism of homology search expansion during recombinational DNA break repair in Saccharomyces cerevisiae.

Homology search is a central step of DNA double-strand break (DSB) repair by homologous recombination (HR). How it operates in cells remains elusive. We developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from local to genome-wide search by increasing the probability of assembling extensive NPFs. DSB end-tethering promotes coordinated search by opposite NPFs. Finally, an autonomous genetic element on chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals the mechanism of the progressive expansion of homology search that is orchestrated by chromatin organizers, long-range resection, end-tethering, and specialized genetic elements and that exploits the stiff NPF structure conferred by Rad51 oligomerization.

Single Nucleotide Polymorphisms in APE1, hOGG1, RAD51 Genes and their Association with Radiotherapy Induced Toxicity among Head and Neck Cancer Patients.

BACKGROUND: Radiotherapy (RT) is a crucial treatment for head and neck cancer however, it causes adverse reactions to the normal tissue and organs adjacent to target tumor. The present study was carried out to investigate possible association of single nucleotide polymorphism in DNA repair genes with toxicity effects of radiotherapy on normal tissue. METHODS: Three hundred and fifty head and neck cancer patients receiving radiotherapy treatment were enrolled in this study. The adverse after effects of radiotherapy on the normal tissue in the form of skin reactions were recorded. Single nucleotide polymorphisms of APE1 (rs1130409), hOGG1 (rs1052133) and Rad51 (rs1801320, rs1801321) genes were studied by polymerase chain reaction-Restriction fragment length polymorphism (PCR-RFLP) and direct DNA sequencing methods and their association with development of severe radio-toxicity effects was evaluated logistic regression analysis. RESULTS: The 172G/T polymorphism of Rad51 was 2.85 times higher and significantly associated with skin reactions (OR=2.85, 95% CI: 1.50-5.41; p=0.001) and severe oral mucositis (OR=4.96, 95% CI: 2.40-10.25; p<0.0001). These results suggested that the polymorphic nature of Rad51 is responsible for risk of radiotherapy adverse effects in HNC patients. The variant 326Cys and heterozygous 326Ser/Cys genotype of hOGG1 was significantly associated with high tumor grade (OR=3.16 95% CI: 1.66-5.99; p=0.0004, and OR=3.97 95% CI: 2.15-7.34; p=<0.0001 respectively). The homozygous variant 172TT genotype of Rad51 showed positive association with poor response of both tumor and nodes towards radiotherapy treatment (p=0.007 and p=0.022). CONCLUSIONS: Interpretation of our results revealed significant association of rs1801321 SNP of Rad51 with development of adverse toxicity reactions in normal tissue of head and neck cancer patients treated with radiotherapy.

The role of SLFN11 in DNA replication stress response and its implications for the Fanconi anemia pathway.

Fanconi anemia (FA) is a hereditary disorder characterized by a deficiency in the repair of DNA interstrand crosslinks and the response to replication stress. Endogenous DNA damage, most likely caused by aldehydes, severely affects hematopoietic stem cells in FA, resulting in progressive bone marrow failure and the development of leukemia. Recent studies revealed that expression levels of SLFN11 affect the replication stress response and are a strong determinant in cell killing by DNA-damaging cancer chemotherapy. Because SLFN11 is highly expressed in the hematopoietic system, we speculated that SLFN11 may have a significant role in FA pathophysiology. Indeed, we found that DNA damage sensitivity in FA cells is significantly mitigated by the loss of SLFN11 expression. Mechanistically, we demonstrated that SLFN11 destabilizes the nascent DNA strands upon replication fork stalling. In this review, we summarize our work regarding an interplay between SLFN11 and the FA pathway, and the role of SLFN11 in the response to replication stress.

CDK phosphorylation of Sfr1 downregulates Rad51 function in late-meiotic homolog invasions.

Meiosis is the developmental program that generates gametes. To produce healthy gametes, meiotic recombination creates reciprocal exchanges between each pair of homologous chromosomes that facilitate faithful chromosome segregation. Using fission yeast and biochemical, genetic, and cytological approaches, we have studied the role of CDK (cyclin-dependent kinase) in the control of Swi5-Sfr1, a Rad51-recombinase auxiliary factor involved in homolog invasion during recombination. We show that Sfr1 is a CDK target, and its phosphorylation downregulates Swi5-Sfr1 function in the meiotic prophase. Expression of a phospho-mimetic sfr1-7D mutant inhibits Rad51 binding, its robust chromosome loading, and subsequently decreases interhomolog recombination. On the other hand, the non-phosphorylatable sfr1-7A mutant alters Rad51 dynamics at late prophase, and exacerbates chromatin segregation defects and Rad51 retention observed in dbl2 deletion mutants when combined with them. We propose Sfr1 phospho-inhibition as a novel cell-cycle-dependent mechanism, which ensures timely resolution of recombination intermediates and successful chromosome distribution into the gametes. Furthermore, the N-terminal disordered part of Sfr1, an evolutionarily conserved feature, serves as a regulatory platform coordinating this phospho-regulation, protein localization and stability, with several CDK sites and regulatory sequences being conserved.

Metformin combined with cisplatin reduces anticancer activity via ATM/CHK2-dependent upregulation of Rad51 pathway in ovarian cancer.

Ovarian cancer (OC) is the deadliest malignancy of the female reproductive system. The standard first-line therapy for OC involves cytoreductive surgical debulking followed by chemotherapy based on platinum and paclitaxel. Despite these treatments, there remains a high rate of tumor recurrence and resistance to platinum. Recent studies have highlighted the potential anti-tumor properties of metformin (met), a traditional diabetes drug. In our study, we investigated the impact of met on the anticancer activities of cisplatin (cDDP) both in vitro and in vivo. Our findings revealed that combining met with cisplatin significantly reduced apoptosis in OC cells, decreased DNA damage, and induced resistance to cDDP. Furthermore, our mechanistic study indicated that the resistance induced by met is primarily driven by the inhibition of the ATM/CHK2 pathway and the upregulation of the Rad51 protein. Using an ATM inhibitor, KU55933, effectively reversed the cisplatin resistance phenotype. In conclusion, our results suggest that met can antagonize the effects of cDDP in specific types of OC cells, leading to a reduction in the chemotherapeutic efficacy of cDDP.

Four cancer cases with pathological germline variant RAD51D c.270_271dup.

Pathological germline variants (PGVs) of RAD51D increase the risk of breast and ovarian cancer. In East Asia, c.270_271dup is the most frequently detected PGV of RAD51D; however, only a few cases have been reported in Japan. We report four cancer cases with a germline RAD51D c.270_271dup PGV. Three of them (lung cancer: 2, oral cancer: 1) were incidentally identified by whole genome sequencing in patients negative for the associated cancer histories, homologous recombination (HR) deficiency, or a second hit of RAD51D in the cancer DNA. For genetic counseling, we provided information on surveillance and cascade testing based on Western guidelines. The PGVs of moderate-risk HR-related genes are difficult to detect based on phenotype, especially in male-predominant pedigrees. The current spread of cancer genomic analysis will increase opportunities for incidental variant identification. The establishment of Japanese guidelines is expected to aid in the management of PGV carriers of moderate-risk genes.

Induction of homologous recombination by site-specific replication stress.

DNA replication stress is one of the primary causes of genome instability. In response to replication stress, cells can employ replication restart mechanisms that rely on homologous recombination to resume replication fork progression and preserve genome integrity. In this review, we provide an overview of various methods that have been developed to induce site-specific replication fork stalling or collapse in eukaryotic cells. In particular, we highlight recent studies of mechanisms of replication-associated recombination resulting from site-specific protein-DNA barriers and single-strand breaks, and we discuss the contributions of these findings to our understanding of the consequences of these forms of stress on genome stability.

Synergistic combination of RAD51-SCR7 improves CRISPR-Cas9 genome editing efficiency by preventing R-loop accumulation.

CRISPR-Cas9 has emerged as a powerful tool for genome editing. However, Cas9 genome editing faces challenges, including low efficiency and off-target effects. Here, we report that combined treatment with RAD51, a key factor in homologous recombination, and SCR7, a DNA ligase IV small-molecule inhibitor, enhances CRISPR-Cas9-mediated genome-editing efficiency in human embryonic kidney 293T and human induced pluripotent stem cells, as confirmed by cyro- transmission electron microscopy and functional analyses. First, our findings reveal the crucial role of RAD51 in homologous recombination (HR)-mediated DNA repair process. Elevated levels of exogenous RAD51 promote a post-replication step via single-strand DNA gap repair process, ensuring the completion of DNA replication. Second, using the all-in-one CRISPR-Cas9-RAD51 system, highly expressed RAD51 improved the multiple endogenous gene knockin/knockout efficiency and insertion/deletion (InDel) mutation by activating the HR-based repair pathway in concert with SCR7. Sanger sequencing shows distinct outcomes for RAD51-SCR7 in the ratio of InDel mutations in multiple genome sites. Third, RAD51-SCR7 combination can induce efficient R-loop resolution and DNA repair by enhanced HR process, which leads to DNA replication stalling and thus is advantageous to CRISPR-Cas9-based stable genome editing. Our study suggests promising applications in genome editing by enhancing CRISPR-Cas9 efficiency through RAD51 and SCR7, offering potential advancements in biotechnology and therapeutics.