Processing and Visualization of Protec-Seq Data for the Generation of Calibrated, Genome-Wide Double Double-Strand Break (dDSB) Maps.

This chapter describes the computational pipeline for the processing and visualization of Protec-Seq data, a method for purification and genome-wide mapping of double-stranded DNA protected by a specific protein at both ends. In the published case, the protein of choice was Saccharomyces cerevisiae Spo11, a conserved topoisomerase-like enzyme that makes meiotic double-strand breaks (DSBs) to initiate homologous recombination, ensuring proper segregation of homologous chromosomes and fertility. The isolated DNA molecules were thus termed double DSB (dDSB) fragments and were found to represent 34 to several hundred base-pair long segments that are generated by Spo11 and are enriched at DSB hotspots, which are sites of topological stress. In order to allow quantitative comparisons between dDSB profiles across experiments, we implemented calibrated chromatin immunoprecipitation sequencing (ChIP-Seq) using the meiosis-competent yeast species Saccharomyces kudriavzevii as calibration strain. Here, we provide a detailed description of the computational methods for processing, analyzing, and visualizing Protec-Seq data, comprising the download of the raw data, the calibrated genome-wide alignments, and the scripted creation of either arc plots or Hi-C-style heatmaps for the illustration of chromosomal regions of interest. The workflow is based on Linux shell scripts (including wrappers for publicly available, open-source software) as well as R scripts and is highly customizable through its modular structure.

Exploring the LET dependence of DNA DSB repair kinetics using the DR DNA database.

The repair of DNA double-strand breaks is a crucial yet delicate process which is affected by a multitude of factors. In this study, our goal is to analyse the influence of the linear energy transfer (LET) on the DNA repair kinetics. By utilizing the database of repair of DNA and aggregating the results of 84 experiments, we conduct various model fits to evaluate and compare different hypothesis regarding the effect of LET on the rejoining of DNA ends. Despite the considerable research efforts dedicated to this topic over the past decades, our findings underscore the complexity of the relationship between LET and DNA repair kinetics. This study leverages big data analysis to capture overall trends that single experimental studies might miss, providing a valuable model for understanding how radiation quality impacts DNA damage and subsequent biological effects. Our results highlight the gaps in our current understanding, emphasizing the pressing need for further investigation into this phenomenon.

Impaired DNA Double-Strand Break Repair in Irradiated Sheep Lung Fibroblasts: Late Effects of Previous Irradiation of the Spinal Thecal Sac.

Children with cancer previously treated with radiotherapy face the likelihood of side effects that can be debilitating or fatal. This study aimed to assess the long-term effect of medulloblastoma radiotherapy on the DNA double-strand break (DSB) repair capability of primary fibroblasts derived from lung biopsies of previously irradiated young sheep. This study included biopsies from three control and five irradiated sheep. The treated sheep had previously received spinal radiotherapy at a total dose of 28 Gy, which is equivalent to pediatric medulloblastoma treatment. Lung biopsies were taken 4 years post-irradiation from high-dose (HD, >18 Gy) and low-dose (LD, <2 Gy) regions. Fifteen cell lines were extracted (six control, four LD and five HD). The cells were irradiated, and DNA DSB repair was analyzed by immunofluorescence. Clonogenic, trypan blue and micronuclei assays were performed. Both the HD and LD cell lines had a significantly higher number of residual γH2AX foci 24 h and a significant decrease in pATM activity post-irradiation compared to the control. There was no statistically significant difference in the clonogenic assay, trypan blue and micronuclei results. Our study showed that a previous irradiation can impair the DNA DSB repair mechanism of ovine lung fibroblasts.

The RIG-I-like receptor signaling pathway triggered by Staphylococcus aureus promotes breast cancer metastasis.

Host microbes are increasingly recognized as key components in various types of cancer, although their exact impact remains unclear. This study investigated the functional significance of Staphylococcus aureus (S. aureus) in breast cancer tumorigenesis and progression. We found that S. aureus invasion resulted in a compromised DNA damage response process, as evidenced by the absence of G1-phase arrest and apoptosis in breast cells in the background of double strand breaks production and the activation of the ataxia-telangiectasia mutated (ATM)-p53 signaling pathway. The high-throughput mRNA sequencing, bioinformatics analysis and pharmacological studies revealed that S. aureus facilitates breast cell metastasis through the innate immune pathway, particularly in cancer cells. During metastasis, S. aureus initially induced the expression of RIG-I-like receptors (RIG-I in normal breast cells, RIG-I and MDA5 in breast cancer cells), which in turn activated NF-κB p65 expression. We further showed that NF-κB p65 activated the CCL5-CCR5 pathway, contributing to breast cell metastasis. Our study provides novel evidence that the innate immune system, triggered by bacterial infection, plays a role in bacterial-driven cancer metastasis.

Differential contributions of double-strand break repair pathways to DNA rearrangements following the irradiation of Arabidopsis seeds and seedlings with ion beams.

DNA rearrangements, including inversions, translocations, and large insertions/deletions (indels), are crucial for crop evolution, domestication, and improvement. The rearrangements are frequently induced by ion beams via the mis-repair of DNA double-strand breaks (DSBs). Unfortunately, how ion beam-induced DSBs are repaired has not been comprehensively analyzed and the mechanisms underlying DNA rearrangements remain unclear. In this study, clonal sectors originating from single mutated cells in carbon ion-irradiated plants were used for whole-genome sequencing analyses after Arabidopsis seeds and seedlings were irradiated. Comparative analyses of the induced mutations (e.g., size and frequency of indels and microhomology at the junctions of the rearrangements) in the irradiated materials suggested that the broken/rejoined DSB ends were more extensively processed in seedlings than in seeds. A mutation to canonical non-homologous end-joining (c-NHEJ), which is a DSB repair pathway with minimal processing of DSB ends, increased the sensitivity to ion beams more in the seeds than in the seedlings, which was consistent with the junction analysis results, indicative of the minor contribution of c-NHEJ to the carbon ion-induced DSB repair in seedlings. Considering the characteristics of the large templated insertions in irradiated seedlings, ion-beam-induced DSBs in seedlings are likely repaired primarily by a polymerase theta-mediated pathway. Polymerase theta-deficient seedlings were more sensitive to ion beams than the c-NHEJ-deficient seedlings, consistent with this hypothesis. This study revealed the key characteristics of ion beam-induced DSBs and the associated repair mechanisms related to the physiological status of the irradiated materials, with implications for elucidating the occurrence and induction of rearrangements.

Early and Late Effects of Low-Dose X-ray Exposure in Human Fibroblasts: DNA Repair Foci, Proliferation, Autophagy, and Senescence.

The effects of low-dose radiation exposure remain a controversial topic in radiation biology. This study compares early (0.5, 4, 24, 48, and 72 h) and late (5, 10, and 15 cell passages) post-irradiation changes in γH2AX, 53BP1, pATM, and p-p53 (Ser-15) foci, proliferation, autophagy, and senescence in primary fibroblasts exposed to 100 and 2000 mGy X-ray radiation. The results show that exposure to 100 mGy significantly increased γH2AX, 53BP1, and pATM foci only at 0.5 and 4 h post irradiation. There were no changes in p-p53 (Ser-15) foci, proliferation, autophagy, or senescence up to 15 passages post irradiation at the low dose.

The Arp2/3 inhibitory protein Arpin inhibits homology-directed DNA repair.

BACKGROUND INFORMATION: Arpin, an Arp2/3 inhibitory protein, inhibits lamellipodial protrusions and cell migration. Arpin expression is lost in tumor cells of several cancer types. RESULTS: Here we analyzed expression levels of Arpin and various markers using Reverse Phase Protein Array (RPPA) in human mammary carcinomas. We found that Arpin protein levels were correlated with those of several DNA damage response markers. Arpin-null cells display enhanced clustering of double stand breaks (DSBs) when cells are treated with a DNA damaging agent, in line with a previously described role of the Arp2/3 complex in promoting DSB clustering for homologous DNA repair (HDR) in the nucleus. Using a specific HDR assay, we further showed that Arpin depletion increased HDR efficiency two-fold through its ability to inactivate the Arp2/3 complex. CONCLUSIONS: Arpin regulates both cell migration in the cytosol and HDR in the nucleus. SIGNIFICANCE: Loss of Arpin expression coordinates enhanced cell migration with up-regulated DNA repair, which is required when DNA damage is induced by active cell migration.

Talazoparib enhances resection at DSBs and renders HR-proficient cancer cells susceptible to Polθ inhibition.

BACKGROUND AND PURPOSE: The PARP inhibitor (PARPi), Talazoparib (BMN673), effectively and specifically radiosensitizes cancer cells. Radiosensitization is mediated by a shift in the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) toward PARP1-independent, alternative end-joining (alt-EJ). DNA polymerase theta (Polθ) is a key component of this PARP1-independent alt-EJ pathway and we show here that its inhibition can further radiosensitize talazoparib-treated cells. The purpose of the present work is to explore mechanisms and dynamics underpinning enhanced talazoparib radiosensitization by Polθ inhibitors in HR-proficient cancer cells. METHODS AND MATERIALS: Radiosensitization to PARPis, talazoparib, olaparib, rucaparib and veliparib was assessed by clonogenic survival. Polθ-proficient and -deficient cells were treated with PARPis and/or with the Polθ inhibitors ART558 or novobiocin. The role of DNA end-resection was studied by down-regulating CtIP and MRE11 expression using siRNAs. DSB repair was assessed by scoring γH2AX foci. The formation of chromosomal abnormalities was assessed as evidence of alt-EJ function using G2-specific cytogenetic analysis. RESULTS: Talazoparib exerted pronounced radiosensitization that varied among the tested cancer cell lines; however, radiosensitization was undetectable in normal cells. Other commonly used PARPis, olaparib, veliparib, or rucaparib were ineffective radiosensitizers under our experimental conditions. Although genetic ablation or pharmacological inhibition of Polθ only mildly radiosensitized cancer cells, talazoparib-treated cells were markedly further radiosensitized. Mechanistically, talazoparib shunted DSBs to Polθ-dependent alt-EJ by enhancing DNA end-resection in a CtIP- and MRE11-dependent manner - an effect detectable at low, but not high IR doses. Chromosomal translocation analysis in talazoparib-treated cells exposed to Polθ inhibitors suggested that PARP1- and Polθ-dependent alt-EJ pathways may complement, but also back up each other. CONCLUSION: We propose that talazoparib promotes low-dose, CtIP/MRE11-dependent resection and increases the reliance of irradiated HR-proficient cancer cells, on Polθ-mediated alt-EJ. The combination of Polθ inhibitors with talazoparib suppresses this option and causes further radiosensitization. The results suggest that Polθ inhibition may be exploited to maximize talazoparib radiosensitization of HR-proficient tumors in the clinic.

Fance deficiency impaired DNA damage repair of prospermatogonia and altered the repair dynamics of spermatocytes.

BACKGROUND: Non-obstructive azoospermia (NOA) is the most severe form of male infertility and affects approximately 1% of men worldwide. Fanconi anemia (FA) genes were known for their essential role in DNA repair and growing evidence showed the crucial role of FA pathway in NOA. However, the underlying mechanisms for Fance deficiency lead to a serious deficit and delayed maturation of male germ cells remain unclear. METHODS: We used Fance deficiency mouse model for experiments, and collected testes or epididymides from mice at 8 weeks (8W), 17.5 days post coitum (dpc), and postnatal 11 (P11) to P23. The mice referred to three genotypes: wildtype (Fance +/+), heterozygous (Fance +/-), and homozygous (Fance -/-). Hematoxylin and eosin staining, immunofluorescence staining, and surface spread of spermatocytes were performed to explore the mechanisms for NOA of Fance -/- mice. Each experiment was conducted with a minimum of three biological replicates and Kruskal-Wallis with Dunn's correction was used for statistical analysis. RESULTS: In the present study, we found that the adult male Fance -/- mice exhibited massive germ cell loss in seminiferous tubules and dramatically decreased sperms in epididymides. During the embryonic period, the number of Fance -/- prospermatogonia decreased significantly, without impacts on the proliferation (Ki-67, PCNA) and apoptosis (cleaved PARP, cleaved Caspase 3) status. The DNA double-strand breaks (γH2AX) increased at the cellular level of Fance -/- prospermatogonia, potentially associated with the increased nonhomologous end joining (53BP1) and decreased homologous recombination (RAD51) activity. Besides, Fance deficiency impeded the progression of meiotic prophase I of spermatocytes. The mechanisms entailed the reduced recruitment of the DNA end resection protein RPA2 at leptotene and recombinases RAD51 and DMC1 at zygotene. It also involved impaired removal of RPA2 at zygotene and FANCD2 foci at pachytene. And the accelerated initial formation of crossover at early pachytene, which is indicated by MLH1. CONCLUSIONS: Fance deficiency caused massive male germ cell loss involved in the imbalance of DNA damage repair in prospermatogonia and altered dynamics of proteins in homologous recombination, DNA end resection, and crossover, providing new insights into the etiology and molecular basis of NOA.

Deficiency in epithelium RAD50 aggravates UC via IL-6-mediated JAK1/2-STAT3 signaling and promotes development of colitis-associated cancer in mice.

BACKGROUND: Ulcerative colitis (UC) is one of the most important risk factors for developing colitis-associated cancer (CAC). Persistent DNA damage increases CAC risk and has been observed in patients with UC. We aimed to identify the regulatory role of RAD50, a DNA double-strand breaks (DSBs) sensor, in UC progression to CAC. METHODS: DSBs and RAD50 expression in IBD and CAC cell and mouse models were assessed. Mice with intestinal epithelial RAD50 deletion (RAD50IEC-KO) were used to examine the role of RAD50 in colitis and CAC. RESULTS: Along with the increased γ-H2AX expression in colitis and CAC models, RAD50 expression reduced in human IBD and CAC as well as in mouse models. Furthermore, RAD50IEC-KO sensitizes mice to dextran sulfate sodium (DSS)-induced acute and chronic experimental colitis. RNA-seq analyses revealed that RAD50 activated the cytokine-cytokine receptor response, which was amplified through the JAK-STAT pathway. RAD50 directly interacts with STAT3 and subsequently inhibits its phosphorylation, which may disrupt the IL-6-JAK1/2-STAT3-IL-6 feed-forward loop. Pharmacological STAT3 inhibition relieves colitis in RAD50IEC-KO mice. Severe DSBs, increased cell proliferation, and extended inflammatory response were identified in RAD50-deficienct cells, which promoted azoxymethane (AOM)-DSS-induced colon tumor development in RAD50IEC-KO mice. CONCLUSION: RAD50 exerts anti-IL-6-related inflammatory effects in colitis and suppresses CAC. Increasing RAD50 level in colon tissues may be promising for treating patients with UC and CAC.

Accidental Encounter of Repair Intermediates in Alkylated DNA May Lead to Double-Strand Breaks in Resting Cells.

In clinics, chemotherapy is often combined with surgery and radiation to increase the chances of curing cancers. In the case of glioblastoma (GBM), patients are treated with a combination of radiotherapy and TMZ over several weeks. Despite its common use, the mechanism of action of the alkylating agent TMZ has not been well understood when it comes to its cytotoxic effects in tumor cells that are mostly non-dividing. The cellular response to alkylating DNA damage is operated by an intricate protein network involving multiple DNA repair pathways and numerous checkpoint proteins that are dependent on the type of DNA lesion, the cell type, and the cellular proliferation state. Among the various alkylating damages, researchers have placed a special on O6-methylguanine (O6-mG). Indeed, this lesion is efficiently removed via direct reversal by O6-methylguanine-DNA methyltransferase (MGMT). As the level of MGMT expression was found to be directly correlated with TMZ efficiency, O6-mG was identified as the critical lesion for TMZ mode of action. Initially, the mode of action of TMZ was proposed as follows: when left on the genome, O6-mG lesions form O6-mG: T mispairs during replication as T is preferentially mis-inserted across O6-mG. These O6-mG: T mispairs are recognized and tentatively repaired by a post-replicative mismatched DNA correction system (i.e., the MMR system). There are two models (futile cycle and direct signaling models) to account for the cytotoxic effects of the O6-mG lesions, both depending upon the functional MMR system in replicating cells. Alternatively, to explain the cytotoxic effects of alkylating agents in non-replicating cells, we have proposed a "repair accident model" whose molecular mechanism is dependent upon crosstalk between the MMR and the base excision repair (BER) systems. The accidental encounter between these two repair systems will cause the formation of cytotoxic DNA double-strand breaks (DSBs). In this review, we summarize these non-exclusive models to explain the cytotoxic effects of alkylating agents and discuss potential strategies to improve the clinical use of alkylating agents.

BRCA1/BRC-1 and SMC-5/6 regulate DNA repair pathway engagement during Caenorhabditis elegans meiosis.

The preservation of genome integrity during sperm and egg development is vital for reproductive success. During meiosis, the tumor suppressor BRCA1/BRC-1 and structural maintenance of chromosomes 5/6 (SMC-5/6) complex genetically interact to promote high fidelity DNA double strand break (DSB) repair, but the specific DSB repair outcomes these proteins regulate remain unknown. Using genetic and cytological methods to monitor resolution of DSBs with different repair partners in Caenorhabditis elegans, we demonstrate that both BRC-1 and SMC-5 repress intersister crossover recombination events. Sequencing analysis of conversion tracts from homolog-independent DSB repair events further indicates that BRC-1 regulates intersister/intrachromatid noncrossover conversion tract length. Moreover, we find that BRC-1 specifically inhibits error prone repair of DSBs induced at mid-pachytene. Finally, we reveal functional interactions of BRC-1 and SMC-5/6 in regulating repair pathway engagement: BRC-1 is required for localization of recombinase proteins to DSBs in smc-5 mutants and enhances DSB repair defects in smc-5 mutants by repressing theta-mediated end joining (TMEJ). These results are consistent with a model in which some functions of BRC-1 act upstream of SMC-5/6 to promote recombination and inhibit error-prone DSB repair, while SMC-5/6 acts downstream of BRC-1 to regulate the formation or resolution of recombination intermediates. Taken together, our study illuminates the coordinated interplay of BRC-1 and SMC-5/6 to regulate DSB repair outcomes in the germline.

Induction of DNA single- and double-strand breaks by excited intra- or extracellular green fluorescent protein.

Green fluorescent protein (GFP) has opened vast new avenues in studies of live cells and is generally perceived as a benign, nontoxic and harmless fluorescent tag. We demonstrat that excited GFP is capable of inducing substantial DNA damage in cells expressing fusion proteins. In the presence of GFP, even low doses of blue light (12 µJ) induce single strand breaks (SSBs). When the fluorescence of GFP located in the cell nucleus or in the cytoplasm is excited by a much higher dose (17 mJ), DNA double-strand breaks (DSBs) are also induced. Such breaks are induced even when GFP is placed and illuminated in culture medium outside of living cells. We demonstrate that DNA damage is induced by singlet oxygen, which is generated by excited GFP. Although short exposures of live cells to exciting light typically used in fluorescence microscopy induce SSBs but carry little risk of inducing DNA double-strand breaks, larger doses, which may be used in FRAP, FLIM, FCS and super-resolution fluorescence microscopy studies, are capable of inducing not only numerous SSBs but also DSBs.

Profiling Cas9- and Cas12a-induced mutagenesis in Arabidopsis thaliana.

With the advancement of CRISPR technologies, a comprehensive understanding of repair mechanisms following double-strand break (DSB) formation is important for improving the precision and efficiency of genetic modifications. In plant genetics, two Cas nucleases are widely used, i.e. Cas9 and Cas12a, which differ with respect to PAM sequence composition, position of the DSB relative to the PAM, and DSB-end configuration (blunt vs. staggered). The latter difference has led to speculations about different options for repair and recombination. Here, we provide detailed repair profiles for LbCas12a in Arabidopsis thaliana, using identical experimental settings previously reported for Cas9-induced DSBs, thus allowing for a quantitative comparison of both nucleases. For both enzymes, non-homologous end-joining (NHEJ) produces 70% of mutations, whereas polymerase theta-mediated end-joining (TMEJ) generates 30%, indicating that DSB-end configuration does not dictate repair pathway choice. Relevant for genome engineering approaches aimed at integrating exogenous DNA, we found that Cas12a similarly stimulates the integration of T-DNA molecules as does Cas9. Long-read sequencing of both Cas9 and Cas12a repair outcomes further revealed a previously underappreciated degree of DNA loss upon TMEJ. The most notable disparity between Cas9 and Cas12a repair profiles is caused by how NHEJ acts on DSB ends with short overhangs: non-symmetric Cas9 cleavage produce 1 bp insertions, which we here show to depend on polymerase Lambda, whereas staggered Cas12a DSBs are not subjected to fill-in synthesis. We conclude that Cas9 and Cas12a are equally effective for genome engineering purposes, offering flexibility in nuclease choice based on the availability of compatible PAM sequences.

DSB profiles in human spermatozoa highlight the role of TMEJ in the male germline.

The male mammalian germline is characterized by substantial chromatin remodeling associated with the transition from histones to protamines during spermatogenesis, followed by the reversal to nucleohistones in the male pronucleus preceding the zygotic genome activation. Both transitions are associated with the extensive formation of DNA double-strand breaks (DSBs), requiring an estimated 5 to 10 million transient DSBs per spermatozoa. Additionally, the high transcription rate in early stages of spermatogenesis leads to transcription-coupled damage preceding meiotic homologous recombination, potentially further contributing to the DSB landscape in mature spermatozoa. Once meiosis is completed, spermatozoa remain haploid and therefore cannot rely on error-free homologous recombination, but instead depend on error-prone classical non-homologous end joining (cNHEJ). This DNA damage/repair-scenario is proposed to be one of the main causes of the observed paternal mutation propensity in human evolution. Recent studies have shown that DSBs in the male pronucleus are repaired by maternally provided Polθ in Caenorhabditis elegans through Polθ-mediated end joining (TMEJ). Additionally, population genetic datasets have revealed a preponderance of TMEJ signatures associated with human variation. Since these signatures are the result of the combined effect of TMEJ and DSB formation in spermatozoa and male pronuclei, we used a BLISS-based protocol to analyze recurrent DSBs in mature human sperm heads as a proxy of the male pronucleus before zygotic chromatin remodeling. The DSBs were found to be enriched in (YR)n short tandem repeats and in evolutionarily young SINEs, reminiscent to patterns observed in murine spermatids, indicating evolutionary hotspots of recurrent DSB formation in mammalian spermatozoa. Additionally, we detected a similar DSB pattern in diploid human IMR90 cells when cNHEJ was selectively inhibited, indicating the significant impact of absent cNHEJ on the sperm DSB landscape. Strikingly, regions associated with most retained histones, and therefore less condensed chromatin, were not strongly enriched with recurrent DSBs. In contrast, the fraction of retained H3K27me3 in the mature spermatozoa displayed a strong association with recurrent DSBs. DSBs in H3K27me3 are associated with a preference for TMEJ over cNHEJ during repair. We hypothesize that the retained H3K27me3 may trigger transgenerational DNA repair by priming maternal Polθ to these regions.

In vitro validation of helium ion irradiations as a function of linear energy transfer in radioresistant human malignant cells.

PURPOSE: Based on considerable interest to enlarge the experimental database of radioresistant cells after their irradiation with helium ions, HTB140, MCF-7 and HTB177 human malignant cells are exposed to helium ion beams having different linear energy transfer (LET). MATERIALS AND METHODS: The cells are irradiated along the widened 62 MeV/u helium ion Bragg peak, providing LET of 4.9, 9.8, 23.4 and 36.8 keV/µm. Numerical simulations with the Geant4 toolkit are used for the experimental design. Cell survival is evaluated and compared with reference γ-rays. DNA double strand breaks are assessed via γ-H2AX foci. RESULTS: With the increase of LET, surviving fractions at 2 Gy decrease, while RBE (2 Gy, γ) gradually increase. For HTB140 cells, above the dose of 4 Gy, a slight saturation of survival is observed while the increase of RBE (2 Gy, γ) remains unaffected. With the increase of LET the increase of γ-H2AX foci is revealed at 0.5 h after irradiation. There is no significant difference in the number of foci between the cell lines for the same LET. From 0.5 to 24 h, the number of foci drops reaching its residual level. For each time point, there are small differences in DNA DSB among the three cell lines. CONCLUSION: Analyses of data acquired for the three cell lines irradiated by helium ions, having different LET, reveal high elimination capacity and creation of a large number of DNA DSB with respect to γ-rays, and are between those reported for protons and carbon ions.

Low testing rates and high BRCA prevalence: Poly (ADP-ribose) polymerase inhibitor use in Middle East BRCA/homologous recombination deficiency-positive cancer patients.

BACKGROUND: Poly (ADP-ribose) polymerase inhibitors (PARPis) are approved as first-line therapies for breast cancer gene (BRCA)-positive, human epidermal growth factor receptor 2-negative locally advanced or metastatic breast cancer. They are also effective for new and recurrent ovarian cancers that are BRCA- or homologous recombination deficiency (HRD)-positive. However, data on these mutations and PARPi use in the Middle East are limited. AIM: To assess BRCA/HRD prevalence and PARPi use in patients in the Middle East with breast/ovarian cancer. METHODS: This was a single-center retrospective study of 57 of 472 breast cancer patients tested for BRCA mutations, and 25 of 65 ovarian cancer patients tested for HRD. These adult patients participated in at least four visits to the oncology service at our center between August 2021 and May 2023. Data were summarized using descriptive statistics and compared using counts and percentages. Response to treatment was assessed using Response Evaluation Criteria in Solid Tumors criteria. RESULTS: Among the 472 breast cancer patients, 12.1% underwent BRCA testing, and 38.5% of 65 ovarian cancer patients received HRD testing. Pathogenic mutations were found in 25.6% of the tested patients: 26.3% breast cancers had germline BRCA (gBRCA) mutations and 24.0% ovarian cancers showed HRD. Notably, 40.0% of gBRCA-positive breast cancers and 66.0% of HRD-positive ovarian cancers were Middle Eastern and Asian patients, respectively. PARPi treatment was used in 5 (33.3%) gBRCA-positive breast cancer patients as first-line therapy (n = 1; 7-months progression-free), for maintenance (n = 2; > 15-months progression-free), or at later stages due to compliance issues (n = 2). Four patients (66.6%) with HRD-positive ovarian cancer received PARPi and all remained progression-free. CONCLUSION: Lower testing rates but higher BRCA mutations in breast cancer were found. Ethnicity reflected United Arab Emirates demographics, with breast cancer in Middle Eastern and ovarian cancer in Asian patients.

CX-5461 Preferentially Induces Top2α-Dependent DNA Breaks at Ribosomal DNA Loci.

While genotoxic chemotherapeutic agents are among the most effective tools to combat cancer, they are often associated with severe adverse effects caused by indiscriminate DNA damage in non-tumor tissue as well as increased risk of secondary carcinogenesis. This study builds on our previous work demonstrating that the RNA Polymerase I (Pol I) transcription inhibitor CX-5461 elicits a non-canonical DNA damage response and our discovery of a critical role for Topoisomerase 2α (Top2α) in the initiation of Pol I-dependent transcription. Here, we identify Top2α as a mediator of CX-5461 response in the murine Eµ-Myc B lymphoma model whereby sensitivity to CX-5461 is dependent on cellular Top2α expression/activity. Most strikingly, and in contrast to canonical Top2α poisons, we found that the Top2α-dependent DNA damage induced by CX-5461 is preferentially localized at the ribosomal DNA (rDNA) promoter region, thereby highlighting CX-5461 as a loci-specific DNA damaging agent. This mechanism underpins the efficacy of CX-5461 against certain types of cancer and can be used to develop effective non-genotoxic anticancer drugs.

TDP43 interacts with MLH1 and MSH6 proteins in a DNA damage-inducible manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double-strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that the TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complementary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 h treatment of 10 µM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS-affected neurons.

The Influence of Homologous Arm Length on Homologous Recombination Gene Editing Efficiency Mediated by SSB/CRISPR-Cas9 in Escherichia coli.

The precise editing of genes mediated by CRISPR-Cas9 necessitates the application of donor DNA with appropriate lengths of homologous arms and fragment sizes. Our previous development, SSB/CRISPR-Cas9, has demonstrated high efficiency in homologous recombination and non-homologous end joining gene editing within bacteria. In this study, we optimized the lengths and sizes of homologous arms of the donor DNA within this system. Two sets of donor DNA constructs were generated: one set comprised donors with only 10-100 bp homologous arms, while the other set included donors with homologous arms ranging from 10-100 bp, between which was a tetracycline resistance expression cassette (1439 bp). These donor constructs were transformed into Escherichia coli MG1655 cells alongside pCas-SSB/pTargetF-lacZ. Notably, when the homologous arms ranged from 10 to 70 bp, the transformation efficiency of non-selectable donors was significantly higher than that of selectable donors. However, within the range of 10-100 bp homologous arm lengths, the homologous recombination rate of selectable donors was significantly higher than that of non-selectable donors, with the gap narrowing as the homologous arm length increased. For selectable donor DNA with homologous arm lengths of 10-60 bp, the homologous recombination rate increased linearly, reaching a plateau when the homologous arm length was between 60-100 bp. Conversely, for non-selectable donor DNA, the homologous recombination rate increased linearly with homologous arm lengths of 10-90 bp, plateauing at 90-100 bp. Editing two loci simultaneously with 100 bp homologous arms, whether selectable or non-selectable, showed no difference in transformation or homologous recombination rates. Editing three loci simultaneously with 100 bp non-selectable homologous arms resulted in a 45% homologous recombination rate. These results suggest that efficient homologous recombination gene editing mediated by SSB/CRISPR-Cas9 can be achieved using donor DNA with 90-100 bp non-selectable homologous arms or 60-100 bp selectable homologous arms.