Homologous Recombination Research Articles

Allelic Variation in gtfB–gtfC Region of Natural Variant of Streptococcus mutans Without Biofilm Formation

Streptococcus mutans is primarily found in biofilms on tooth surfaces and is associated with the development of dental caries. S. mutans synthesizes water-insoluble glucan (WIG) using sucrose as a substrate, inducing the formation of three-dimensional biofilms. WIG is produced by glucosyltransferases (GTFs) encoded by the tandem and highly homologous gtfB and gtfC genes. Conversely, the homologous recombination of gtfB and gtfC readily happens, producing natural variants without WIG. These WIG− variants are thought to have ecologically pleiotropic functions; however, the molecular basis for their appearance is unclear. This study aimed to determine the sequence of the gtfB–gtfC regions of WIG− variants. We sequenced the gtfB–gtfC regions of 23 WIG− variants derived from S. mutans UA159 and revealed the presence of five alleles and four types of single homologous recombination patterns. Regardless of the allele type, the WIG− variants showed low biofilm formation and GTF activity. To the best of our knowledge, this is the first study to report the presence of alleles in WIG− variants. These findings provide important information for explaining the appearance of mechanisms in WIG− variants.

Gene Targeting via CRISPR/Cas9-Mediated DNA Double-Strand Break Induction in Rice.

Gene targeting (GT) is a precise genome editing tool to achieve desired modification of a target gene, e.g., introduction of point mutations, knock-in of a reporter gene, or swapping of a functional domain, through homologous recombination. However, due to its low frequency, it has proved difficult to establish a universal GT system. The availability of the CRISPR/Cas9 system for genome editing in plants has opened up possibilities to apply GT successfully to some plant species. Here, we provide protocols for CRISPR/Cas9-mediated DNA double-strand break (DSB)-induced GT in rice.

DNA damage-induced EMT controlled by the PARP dependent chromatin remodeler ALC1 promotes DNA repair efficiency through RAD51 in tumor cells.

Epithelial-to-mesenchymal transition (EMT) allows cancer cells to metastasize while acquiring resistance to apoptosis and chemotherapeutic agents with significant implications for patients' prognosis and survival. Despite its clinical relevance, the mechanisms initiating EMT during cancer progression remain poorly understood. We demonstrate that DNA damage triggers EMT and that activation of PARP and the PARP-dependent chromatin remodeler ALC1 (CHD1L) was required for this response. Our results suggest that this activation directly facilitates access to the chromatin of EMT transcriptional factors (TFs) which then initiate cell reprogramming. We also show that EMT-TFs bind to the RAD51 promoter to stimulate its expression and to promote DNA repair by homologous recombination (HR). Importantly, a clinically relevant PARP inhibitor reversed or prevented EMT in response to DNA damage while resensitizing tumor cells to other genotoxic agents. Overall, our observations shed light on the intricate relationship between EMT, DNA damage response, and PARP inhibitors, providing potential insights for in cancer therapeutics.

The radiosensitivity of niraparib tosylate to esophageal squamous cell carcinoma is stronger under hypoxia and higher-pressure conditions

Background: Radiotherapy is an effective method for esophageal squamous cell carcinoma (ESCC) treatment show reduced effectiveness in long-term due to reduced sensitivity of cancer cells to radiotherapy. Niraparib tosylate, a poly ADP-ribose polymerase (PARP) inhibitor may enhance the radiosensitivity of these cells. Hence, the present study was aimed to study the effects of niraparib tosylate on ESCC proliferation, apoptosis and cell cycle along with radiosensitivity enhancement efficiency. Materials and methods: ESCC cell lines, KYSE-30 were subjected to niraparib tosylate or irradiation treatments or combination of both. CCK8 and colony formation assays were performed to monitor cell growth status. Cell cycle and apoptosis level were investigated through flow cytometry. The expressions of targeted genes were detected at protein levels by western blot. Besides, xenografts were successfully established in immunocompromised mice. Student’s t-test was used to compare data and P < 0.05 was considered statistically significant. Results: Treatment with niraparib tosylate showed G2/M cycle arrest and apoptosis in KYSE-30 cells and the rate was observed to be more significant with combination therapy. These effects were further strengthened when cells were cultured under hypoxia and high atmospheric pressure. An enhanced susceptibility to radiotherapy was observed with niraparib tosylate combination. The homologous recombination related regulator, Rad51, showed remarkable upregulation in cancer cells exposed to combination of drug-radiation treatment. The tumor burden was also relieved in xenograft mouse models with combination of drug-radiation treatment, indicating the potential of niraparib tosylate use clinical cancer therapy. Conclusion: From the results, a novel function of niraparib tosylate can be established, impairing DNA damage repair efficiency to increase the susceptibility of ESCC to radiotherapy, resulting in cell apoptosis and G2/M cycle arrest in vitro and in vivo.

Origins and impact of extrachromosomal DNA.

Extrachromosomal DNA (ecDNA) is a major contributor to treatment resistance and poor outcome for patients with cancer1,2. Here we examine the diversity of ecDNA elements across cancer, revealing the associated tissue, genetic and mutational contexts. By analysing data from 14,778 patients with39 tumour types from the 100,000 Genomes Project, we demonstrate that 17.1% of tumour samples contain ecDNA. We reveal a pattern highly indicative of tissue-context-based selection for ecDNAs, linking their genomic content to their tissue of origin. We show that not only is ecDNA a mechanism for amplification of driver oncogenes, but it alsoa mechanism that frequently amplifies immunomodulatory and inflammatory genes, such as those that modulate lymphocyte-mediated immunity and immune effector processes. Moreover, ecDNAs carrying immunomodulatory genes are associated with reduced tumour T cell infiltration. We identify ecDNAs bearing only enhancers, promoters and lncRNA elements, suggesting the combinatorial power of interactions between ecDNAs in trans. We alsoidentify intrinsic and environmental mutational processes linked to ecDNA, including those linked to its formation, such as tobacco exposure, and progression, such as homologous recombination repair deficiency. Clinically, ecDNA detection was associated with tumour stage, more prevalent after targeted therapy and cytotoxic treatments, and associated with metastases and shorter overall survival. These results shed light on why ecDNA is a substantial clinical problem that can cooperatively drive tumour growth signals, alter transcriptional landscapes and suppress the immune system.

The Role of Microhomology-Mediated End Joining (MMEJ) at Dysfunctional Telomeres.

DNA double-strand break (DSB) repair pathways are crucial for maintaining genome stability and cell viability. However, these pathways can mistakenly recognize chromosome ends as DNA breaks, leading to adverse outcomes such as telomere fusions and malignant transformation. The shelterin complex protects telomeres from activation of DNA repair pathways by inhibiting nonhomologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ). The focus of this paper is on MMEJ, an error-prone DSB repair pathway characterized by short insertions and deletions flanked by sequence homology. MMEJ is critical in mediating telomere fusions in cells lacking the shelterin complex and at critically short telomeres. Furthermore, studies suggest that MMEJ is the preferred pathway for repairing intratelomeric DSBs and facilitates escape from telomere crisis. Targeting MMEJ to prevent telomere fusions in hematologic malignancies is of potential therapeutic value.

TPX2 serves as a novel target for expanding the utility of PARPi in pancreatic cancer through conferring synthetic lethality

BackgroundPARP inhibitors (PARPi) have been licensed for the maintenance therapy of patients with metastatic pancreatic cancer carrying pathogenic germline BRCA1/2 mutations. However, mutations in BRCA1/2 are notably rare in pancreatic...

Mechanisms of Alternative Lengthening of Telomeres.

In recent years, significant advances have been made in understanding the intricate details of the mechanisms underlying alternative lengthening of telomeres (ALT). Studies of a specialized DNA strand break repair mechanism, known as break-induced replication, and the advent of telomere-specific DNA damaging strategies and proteomic methodologies to profile the ribonucleoprotein composition of telomeres enabled the discovery of networks of proteins that coordinate the stepwise homology-directed DNA repair and DNA synthesis processes of ALT. These networks couple mediators of homologous recombination, DNA template-switching, long-range template-directed DNA synthesis, and DNA strand resolution with SUMO-dependent liquid condensate formation to create discrete nuclear bodies where telomere extension occurs. This review will discuss the recent findings of how these networks may cooperate to mediate telomere extension by the ALT mechanism and their impact on telomere function and integrity in ALT cancer cells.

The Influence of DNA Repair Genes and Prenatal Tobacco Exposure on Risk of Childhood Acute Lymphoblastic Leukemia-A Gene-Environment Interaction Study.

Acute lymphoblastic leukemia (ALL) is the most common type of cancer among children. Tobacco exposure during gestation has been investigated as a potential risk factor, but its role remains undefined. Given tobacco's toxicological profile as a DNA damaging agent, we examined the impact of DNA repair gene variability as a source of vulnerability to tobacco exposure risk for ALL. Leveraging demographic and genotype data from two large California-based ALL epidemiology studies, we used logistic regression, MinimumP (MinP) statistical method and permutation tests to examine interactions between DNA repair genes and prenatal tobacco exposure. We found statistically significant interactions between prenatal tobacco exposure and DNA repair genes RECQL (minP= 1.00x 10-4, FDR-P value = 1.86x 10-2) and TDG (minP= 1.00x 10-4, FDR-P value = 1.86 x 10-2) regarding childhood ALL risk. Notable interactions in the homologous recombination pathway were observed among Latino children, while non-Latino White children displayed significant interactions in the base excision repair and nucleotide excision repair pathways. Our study highlights the significance of DNA repair genes and pathways when evaluating environmental exposure to tobacco smoke, suggesting that genetic variability within these pathways could impact vulnerability in the development of childhood ALL. This study highlights the significant impact of genetic variation interacting with prenatal tobacco exposure on ALL risk. Further research is needed to understand these interactions and their implications for ALL etiology. Expanding studies to other gene-environment interactions will aid in developing targeted prevention, diagnosis, and treatment strategies for pediatric oncology.

TGF-β superfamily-induced transcriptional activation pathways establish the RAD52-dependent ALT machinery during malignant transformation of MPNSTs

To study telomere maintenance mechanism (TMM) activation during malignant transformation, we compared neurofibroma (NF) and malignant peripheral nerve sheath tumor (MPNST) in the same patient with type-1 neurofibromatosis (NF1), a total of 20 NF-MPNST pairs in 20 NF1 patients. These comparisons minimized genetic bias and contrasted only changes associated with malignant transformation, while subtracting changes that developed upon the transformation of normal cells to the benign tumor. TGF-β superfamily genes were found to activate the PAX and SOX transcription factors, leading to TMM activation. BMPER activates PAX6 through BMP2 and PAX7 through BMP4; BMP15 activates SOX14; and INHBC activates PAX9 and SOX14. The activated PAX and SOX genes sequentially establish the core architecture of the RAD52-dependent alternative lengthening of telomeres (ALT). Specifically, PAX7 activates the recombinase (RAD52) and a negative regulator (SLX4IP). PAX6 and SOX14 activate positive regulators (BLM and BRCA2, respectively). PAX9 and SOX14 activate RAD9B and FEN1, which are responsible for the stability of homologous recombination intermediates and increase, together with RAD52, the telomere length. Telomere elongation achieved by the activation of PAX7 and PAX9 is associated with a poor prognosis. We demonstrated that TGF-β superfamily-induced transcriptional activation pathways activated the RAD52-dependent ALT during malignant transformation of MPNSTs.

Transient HR enhancement by RAD51-stimulatory compound confers protection on intestinal rather than hematopoietic tissue against irradiation in mice

DNA double-strand breaks (DSBs) are cytotoxic lesions that compromise genomic integrity and trigger cell death. Homologous recombination (HR) is a major pathway for repairing DSBs in cycling cells. However, it remains unclear whether transient modulation of HR could confer protection to adult stem cells against lethal irradiation exposure. In this study, we investigated the radio-protective effect of the RAD51-stimulatory compound RS-1 on adult stem cells and progenitor cells with varying cycling rates in intestinal and hematopoietic tissues. Treatment with RS-1 even at high doses did not induce noticeable cell death or proliferation of intestinal crypt cells in vivo. Pretreatment with RS-1 before irradiation significantly decreased mitotic death, promoted DNA repair and enhanced the survival of intestinal stem cells and progenitor cells and increased the number of regenerative crypt colonies thereby mitigating IR-induced gastrointestinal syndrome. Moreover, RS1 pretreatment could increase the survival and regeneration of irradiated intestinal organoid in vitro, which can be rescued by RAD51 inhibitor. However, pretreatment with RS-1 in vivo did not elevate nucleated cell count or HSPCs in bone marrow after 6Gy irradiation. Additionally, there was no impact on mouse survival due to drug treatment observed. Thus, our data suggest that targeting HR as a strategy to prevent tissue damage from acute irradiation exposure may depend on cell cycling rates and intrinsic DNA repair mechanisms.

Homologous recombination deficiency score is an independent prognostic factor in esophageal squamous cell carcinoma.

Homologous recombination deficiency (HRD) represents an impairment in the homologous recombination repair (HRR) pathway, crucial for repairing DNA double-strand breaks and contributing to genomic instability in cancer. The HRD score may be a more reliable biomarker than HRR-related gene mutations for identifying patients sensitive to poly(ADP-ribose) polymerase inhibitors. Despite its relevance in various cancers, the HRD score remains underexplored in esophageal squamous cell carcinoma (ESCC). We retrospectively analyzed HRD scores in 96 ESCC patients, examining correlations with clinical characteristics and survival outcomes, and validated our findings using the TCGA dataset. Genomic sequencing utilized a custom superHRD next-generation sequencing panel, and HRD scores were calculated from 54,000 single-nucleotide polymorphisms using Kruskal-Wallis rank-sum tests and two cut-off points for analysis. Higher HRD scores correlated with advanced tumor stages, recurrence, and mutations in TP53 and ABCB1, while APC mutations were linked to lower HRD scores. Patients with high HRD scores had significantly shorter disease-free survival (p = 0.013) and a trend toward shorter overall survival (OS) (p = 0.005), particularly those not receiving adjuvant therapy. Conversely, HRD-high patients undergoing adjuvant therapy showed a trend toward longer OS (p = 0.015). Multivariate analysis identified HRD as an independent prognostic factor (hazard ratio = 2.814 for recurrence, p = 0.015). Validation with the TCGA dataset supported these findings. This study highlights the associations between HRD scores, clinical characteristics, and genomic mutations in ESCC, suggesting HRD as a potential prognostic biomarker. HRD assessment may aid in patient stratification and personalized treatment strategies, warranting further investigation to validate the therapeutic implications of HRD scores in ESCC.

A Gene Expression Signature that Predicts Gastric Cancer Sensitivity to PARP Inhibitor Therapy.

Biomarkers indicating sensitivity to poly ADP-ribose polymerase (PARP) inhibitors have not yet been identified in gastric cancer. PARP inhibitors target homologous recombination deficiency (HRD); however, homologous recombination (HR) induces complex changes in gene expression, which makes it difficult to identify reliable biomarkers. In this study, we identified a multi-gene expression signature as a marker of PARP inhibitor sensitivity in gastric cancer. Seven gastric cancer cell lines were evaluated for susceptibility to PARP inhibitors using a growth inhibition assay. Gene expression profiling (GEP) was used to identify differentially expressed genes between PARP inhibitor-sensitive and -resistant cell lines. The resulting gene set was subjected to cluster analysis using tumor samples from 250 patients who underwent gastrectomy for primary gastroesophageal junction and gastric adenocarcinoma. HRD was defined as a truncating mutation in one or more of 22 HR-related genes and HRD scores were calculated using whole-exome sequencing data. In the growth inhibition assays, the HGC27 and HSC39 cell lines were sensitive to the PARP inhibitors, olaparib, and rucaparib, and were significantly correlated with the GEP results. Seven (2.8%) patients harbored truncating mutations in HR-related genes. A gene expression signature based on the top 100 high and low differentially expressed genes between sensitive and resistant cell lines revealed a patient cluster with a high prevalence of HR-related gene mutations and high HRD scores. The 100-gene expression signature identified in this study may serve as a valuable predictive biomarker for PARP inhibitor sensitivity in gastric cancer.

Construction and characterization of different hemolysin gene deletion strains in Vibrio parahaemolyticus (ΔhlyA, ΔhlyIII) and evaluation of their virulence

Vibrio parahaemolyticus, a halophilic food-borne pathogen, possesses an arsenal of virulence factors. The pathogenicity of V. parahaemolyticus results from a combination of various virulence factors. HlyA and hlyIII genes are presumed to function in hemolysis, in addition to tdh and trh in V. parahaemolyticus. To confirm the hemolytic function of genes hlyA and hlyIII, ΔhlyA and ΔhlyIII strains of V. parahaemolyticus were separately constructed via homologous recombination. The cytotoxicity and pathogenicity of the ΔhlyA and ΔhlyIII strains were evaluated using a Tetrahymena-Vibrio co-culture model and an immersion challenge in Litopenaeus vannamei. Results indicated that the hemolytic activity of the ΔhlyA and ΔhlyIII strains decreased by approximately 31.4 % and 24.9 % respectively, compared to the WT strain. Both ΔhlyA and ΔhlyIII exhibited reduced cytotoxicity towards Tetrahymena. Then shrimp infection experiments showed LD50 values for ΔhlyA and ΔhlyIII of 3.06 × 108 CFU/mL and 1.23 × 108 CFU/mL, respectively, both higher than the WT strain’s value of 2.57 × 107 CFU/mL. Histopathological observations revealed that hepatopancreas from shrimps challenged with ΔhlyA and ΔhlyIII exhibited mild symptoms, whereas those challenged with the WT strain displayed severe AHPND. These findings indicate that the ΔhlyA and ΔhlyIII strains are significantly less virulent than the WT strain. In conclusion, both hlyA and hlyIII are vital virulence genes involved in hemolytic and cytotoxic of V. parahaemolyticus.

Pan-cancer Analysis of Homologous Recombination Deficiency in Cell Lines.

Homologous Recombination Deficiency (HRD) drives genomic instability in multiple cancer types and renders tumors vulnerable to certain DNA damaging agents such as PARP inhibitors. Thus, HRD is emerging as an attractive biomarker in oncology. A variety of in silico methods are available for predicting HRD; however, few of these methods have been applied to cell lines in a comprehensive manner. Here we utilized two of these methods, "CHORD" and "HRDsum" scores, to predict HRD for 1,332 cancer cell lines and 84 non-cancerous cell lines. Cell lines with biallelic mutations in BRCA1 or BRCA2, which encode key components of the homologous recombination pathway, showed the strongest HRD predictions, validating the two methods in cell lines. A small subset of BRCA1/2-wildtype cell lines were also classified as HRD, several of which showed evidence of epigenetic BRCA1 silencing. Similar to HRD in patient samples, HRD in cell lines was associated with p53 loss, was mutually exclusive with microsatellite instability and occurred most frequently in breast and ovarian cancer types. In addition to validating previously identified associations with HRD, we leveraged cell line-specific datasets to gain new insights into HRD and its relation to various genetic dependency and drug sensitivity profiles. We found that in cell lines, HRD was associated with sensitivity to PARP inhibition in breast cancer, but not at a pan-cancer level. By generating large-scale, pan-cancer datasets on HRD predictions in cell lines, we aim to facilitate efforts to improve our understanding of HRD and its utility as a biomarker.

Progression-free and overall survival in newly-diagnosed homologous recombination DNA-repair deficient ovarian cancer without a BRCA mutation

Progression-free and overall survival in newly-diagnosed homologous recombination DNA-repair deficient ovarian cancer without a BRCA mutation

Homologous recombination deficiency scores vary by assay: How can we better harmonize results?

Homologous recombination deficiency scores vary by assay: How can we better harmonize results?

Characterization of epithelial ovarian cancer based on multigene tumor testing and homologous recombination deficiency (HRD) testing (COMBO)

Characterization of epithelial ovarian cancer based on multigene tumor testing and homologous recombination deficiency (HRD) testing (COMBO)

Olaparib monotherapy in advanced triple-negative breast cancer patients with homologous recombination deficiency and without germline mutations in BRCA1/2: the NOBROLA phase 2 study

Olaparib monotherapy in advanced triple-negative breast cancer patients with homologous recombination deficiency and without germline mutations in BRCA1/2: the NOBROLA phase 2 study

Real-world BRCA1/2 and homologous recombination deficiency testing dynamics and time to treatment discontinuation on PARP inhibitor first-line maintenance among patients with epithelial ovarian cancer

Real-world BRCA1/2 and homologous recombination deficiency testing dynamics and time to treatment discontinuation on PARP inhibitor first-line maintenance among patients with epithelial ovarian cancer