Rett syndrome (RTT) and MECP2 duplication syndrome, a subtype of autism spectrum disorder (ASD), are neurodevelopmental disorders caused by MeCP2 loss and gain of function, respectively. While MeCP2 is known to regulate transcription through its interaction with methylated DNA and chromatin-associated factors such as topoisomerase IIβ (TOP2β), the downstream transcriptional consequences of MeCP2 dosage imbalance remain partially characterized. Here, we present a transcriptome-centered analysis of mouse primary cortical neurons subjected to MeCP2 knockdown (KD) or overexpression (OE), which model RTT and ASD-like conditions in parallel. Using a robust computational pipeline integrating generalized linear models with quasi-likelihood F-tests and Magnitude–Altitude Scoring (GLMQL-MAS), we identified differentially expressed genes (DEGs) in KD and OE relative to wild-type (WT) neurons. This study represents a computational analysis of secondary transcriptomic data aimed at nominating candidate genes for future experimental validation. Gene Ontology enrichment revealed both shared and condition-specific biological processes, with KD uniquely affecting neurodevelopmental and stress-response pathways, and OE perturbing extracellular matrix, calcium signaling, and neuroinflammatory processes. To prioritize robust and disease-relevant targets, we applied Cross-MAS and further filtered DEGs by correlation with MeCP2 expression and regulation directional consistency. This yielded 16 high-confidence dosage-sensitive genes that were capable of classifying WT, KD, and OE samples with 100% accuracy using PCA and logistic regression. Among these, RTT-associated candidates such as Plcb1, Gpr161, Mknk2, Rgcc, and Abhd6 were linked to disrupted synaptic signaling and neurogenesis, while ASD-associated genes, including Aim2, Mcm6, Pcdhb9, and Cbs, implicated neuroinflammation and metabolic stress. These findings establish a compact and mechanistically informative set of MeCP2-responsive genes, which enhance our understanding of transcriptional dysregulation in RTT and ASD and nominate molecular markers for future functional validation and therapeutic exploration.

To maintain genomic integrity, cells have evolved several conserved DNA damage response (DDR) pathways in response to DNA damage and stress conditions. Apurinic/apyrimidinic endonuclease 1 (APE1) exhibits AP endonuclease, 3 '-5 ' exonuclease, 3 '-phosphodiesterase, and 3 '-exoribonuclease activities and plays critical roles in the DNA repair and redox regulation of transcription. However, it remains unclear whether and how APE1 is involved in DDR pathways. In this perspective, we first updated our knowledge of APE1's functional domains and its nuclease activities and their specific associated substrates. We then summarized the newly discovered roles and mechanisms of action of APE1 in the global and nucleolar ATR-mediated DDR pathway. While the ATMmediated DDR is well known to be activated by DNA double-strand breaks and oxidative stress, here we provided new perspectives as to how ATM DDR signaling is activated by indirect single-strand breaks (SSBs) resulting from genotoxic stress and defined SSB structures, and discuss how ATM kinase is directly activated and regulated by its activator, APE1. Together, accumulating body of new evidence supports the notion that APE1 is a master regulator protein of the ATR- and ATM-mediated DDR pathways. These new findings of APE1 in DDR signaling provide previously uncharacterized but critical functions and regulations of APE1 in genome integrity.

Abstract Background: Etoposide is a well-characterized chemotherapeutic that promotes DNA double-strand breaks (DSBs) and is associated with therapy-related chromosomal translocations and leukemias. Natural compounds including bioflavonoids, which are found in food as well as in dietary supplements, have been shown by our lab to induce DSBs and promote genome rearrangements. Given the potential for bioflavonoids to cause these harmful effects, we are investigating the potential for a wide array of other natural compounds and environmental agents to also induce DNA DSBs and chromosomal translocations. The purpose of this study was to perform a high throughput screen on a National Institutes of Health (NIH) library of natural compounds to determine their ability to cause DSBs and chromosomal translocations and implicate them as risk factors for leukemias. Methods: We obtained 2 libraires from NIH containing over 2000 natural compounds. Mouse embryonic stem cells (mES) were exposed to individual compounds at 50mM for one hour. Following exposure, DNA damage was measured through γ-H2AX immunocytochemistry and microscopy to quantify the level of DNA damage induced in comparison to etoposide. Compounds identified to cause greater than or equal to 20% of the damage induced by etoposide were prioritized for study of their potential to induce chromosomal translocations. We developed a mES GFP Reporter cell line with engineered exons of GFP contained within MLL and AF9 breakpoint cluster region inserts. Cells were exposed to individual compounds at 50mM for one hour. Following exposure, cells were screened over 7 days for the generation of chromosomal translocation events as measured by appearance of GFP+ cells. Results: Exposure to the NIH natural compound library has thus far generated 22 hits with 5 - 20% the amount of damage caused by etoposide and 29 hits with greater than or equal to 20% of the damage of etoposide out of the 1,200 compounds tested thus far. Top “hits” include known inhibitors of topoisomerase I and II. Other top “hits” include natural compounds curcumin, illudin, prodigiosin, and predorine. Testing of the ability of these “hits” to cause chromosomal translocations are still underway, but compounds, such as curcumin, are showing translocation GFP+ events. Conclusions: Data from this study can be used to inform the public and doctors of natural compounds that may be believed to be beneficial for human health but that can actually cause DNA damage and potential translocations. This data could generate potential drug candidates useful in the treatment of cancer. Etoposide is known to create DSBs but it also causes frequent translocation events. This study could identify compounds that are able to cause DNA DSBs, essential to the destruction of cancer cells, with a lower likelihood of causing chromosomal translocation events in healthy cells. Citation Format: Donna Goodenow, Henry Thompson, Elizabeth Toufekoulas, Heather Derby, Christine Richardson. Identification of novel natural compounds that induce DNA damage and chromosomal translocations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B015.

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function being downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure involves a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune-triggered osmotic changes and actin cytoskeletal remodelling likely driving compact vacuolar morphologies.

Genomic instability is a key driving force for the development and progression of many age-related neurodegenerative diseases and central nervous system (CNS) cancers. Recently, the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), has been shown to detect and respond to self-DNA accumulation resulting from DNA damaging insults in peripheral cell types. cGAS has been shown to be important in the responses of microglia to DNA viruses and amyloid beta, and we have reported that it underlies the responses of human microglia to exogenous DNA. However, the role of this cytosolic sensor in the detection of self-DNA by glia is poorly understood and its ability to mediate the cellular responses of human microglia to genotoxic DNA damage has not been established. Here, we describe the ability of ionizing radiation and oxidative stress to elicit genomic DNA damage in human microglial cells and to stimulate the production of key inflammatory mediators by these cells in an NF-kB dependent manner. Importantly, we have utilized CRISPR/Cas9 and siRNA-mediated knockdown approaches and a pharmacological inhibitor of the cGAS adaptor protein stimulator of interferon genes (STING) to demonstrate that the cGAS-STING pathway plays a critical role in the generation of these microglial immune responses to such genotoxic insults. Together, these studies support the notion that cGAS mediates the detection of cytosolic self-DNA by microglia, providing a potential mechanism linking genomic instability to the development of CNS cancers and neurodegenerative disorders.

Introduction: Ovarian cancer (OC) is the second most common gynecologic cancer in the U.S, and is the deadliest cancer of the female reproductive system. Because of the complex nature of OC, diagnoses are often by display of symptoms at late stages of disease. Identifying OC at early stages can increase the 5-year survival rate from 40% in stage IV to 94% in stage I. Therefore, effective screening and detection of OC at early stages is imperative. The main objective of this study is to develop a diagnostic panel of markers that correlate with early-stage OC, and ultimately, improve the survival rate of patients. Background: Approaches in the past several years to detect malignant OC have focused on processing relevant images or screening genomic data. These studies use liquid-based biopsies, sonographic images, cyst images, CT images, MR imaging and genomics data. Some studies have focused on distinguishing malignant tumors versus benign tumor cells. In addition to statistical methods to examine available genomic datasets, Machine Learning models including K-Nearest Neighbor, K-means, Convolutional Neural Networks, Deep Learning, Support Vector Machine, Random Forest, and Fuzzy algorithms have been used. Methods: In this study we proposed a method to determine a possible correlation between some actively expressed genes and OC, using publicly available mRNA data sets. The samples were retrieved from the GSE106817 dataset via the NCBI GEO site, with 333 cancerous and 2759 Non-cancerous samples. The objective is to define a set of genes which is able to illustrate prediction of correlation through the use of PCA, NMF and SVD. We further verified our findings by testing on one additional different mRNA datasets. For this, we compared several machine learning algorithms to potentially correlate active genes and OC stages, from the available data. Results and Discussions: Through our study we have developed Machine Learning models valuable to identify patterns of disease at different stages, thus, potentially be useful in developing predictive models. Our initial experimental results have been demonstrated with a set of mRNA signatures, which may be used to create a panel of biomarkers valuable for detecting early-stage OC. Our results confirm the correlation between OC and PAX8, PEG3, and BIRC5, and MYB and RAC2. Continuation of this study highlights methods towards the early detection of OC and to improve disease identification outcome. Citation Format: Zahra Saghaie, Christine Richardson, M. Taghi Mostafavi. Early detection of ovarian cancer using mRNA sample data to investigate the correlation of specific gene mutations with ovarian 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 5396.

The nuclear pore complex (NPC) is a large elaborate structure embedded within the nuclear envelope, and intimately linked to the cytoskeleton, nucleoskeleton, and chromatin. Many different cargoes pass through its central channel and along the membrane at its periphery. The NPC is dismantled and reassembly, fully or partially, every cell cycle. In post-mitotic cells it consists of a combination of hyper-stable and highly dynamic proteins. Because of its size, dynamics, heterogeneity and integration, it is not possible to understand its structure and molecular function by any one, or even several, methods. For decades, and to this day, thin section transmission electron microscopy (TEM) has been a central tool for understanding the NPC, its associations, dynamics and role in transport as it can uniquely answer questions concerning fine structural detail within a cellular context. Using immunogold labeling specific components can also be identified within the ultrastructural context. Model organisms such as Saccharomyces cerevisiae are also central to NPC studies but have not been used extensively in structural work. This is because the cell wall presents difficulties with structural preservation and processing for TEM. In recent years, high-pressure freezing and freeze substitution have overcome these problems, as well as opened up methods to combine immunogold labeling with detailed structural analysis. Other model organisms such as the worm Caenorhabditis elegans and the plant Arabidopsis thaliana have been underused for similar reasons, but with similar solutions, which we present here. There are also many advantages to using these methods, adapted for use in mammalian systems, due to the instant nature of the initial fixation, to capture rapid processes such as nuclear transport, and preservation of dynamic membranes.

Maintenance of genome integrity is critical to prevent cell death or disease. Illegitimate repair of chromosomal DNA breaks can lead to mutations and genome rearrangements which are a well-known hallmark of multiple cancers and disorders. Endogenous causes of DNA double-strand breaks (DSBs) include reactive oxygen species (ROS) and replication errors while exogenous causes of DNA breaks include ionizing radiation, UV radiation, alkylating agents, and inhibitors of topoisomerase II (Top2). Recent evidence suggests that a growing list of environmental agents or toxins and natural dietary compounds also cause DNA breaks. Understanding the consequences of exposure to a broad spectrum of DSB-inducing agents has significant implications for understanding mutagenicity, genome stability and human health. This chapter will review in vivo mouse models designed to measure DNA damage and mutagenicity, and illegitimate repair of DNA DSBs caused by exposure to environmental agents.

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. They tether the cell’s plasma membrane (PM) to the actin network. Moreover, in a similar manner, they are also involved in the tethering of membrane bound organelles to the actin cytoskeleton; the endoplasmic reticulum (ER) and the ER to the PM. This raises the question as to whether NET proteins are involved in actin cytoskeletal remodelling. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function as downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure depends on a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune signalling events and proper actin cytoskeletal remodelling.

Epithelial tubes are essential components of metazoan organ systems that control the flow of fluids and the exchange of materials between body compartments and the outside environment. The size and shape of the central lumen confer important characteristics to tubular organs and need to be carefully controlled. Here, we identify the small coiled-coil protein BBLN-1 as a regulator of lumen morphology in the C. elegans intestine. Loss of BBLN-1 causes the formation of bubble-shaped invaginations of the apical membrane into the cytoplasm of intestinal cells and abnormal aggregation of the subapical intermediate filament (IF) network. BBLN-1 interacts with IF proteins and localizes to the IF network in an IF-dependent manner. The appearance of invaginations is a result of the abnormal IF aggregation, indicating a direct role for the IF network in maintaining lumen homeostasis. Finally, we identify bublin (BBLN) as the mammalian ortholog of BBLN-1. When expressed in the C. elegans intestine, BBLN recapitulates the localization pattern of BBLN-1 and can compensate for the loss of BBLN-1 in early larvae. In mouse intestinal organoids, BBLN localizes subapically, together with the IF protein keratin 8. Our results therefore may have implications for understanding the role of IFs in regulating epithelial tube morphology in mammals.

Eukaryotic cells use homologous recombination (HR), classical end-joining (C-NHEJ), and alternative end-joining (Alt-EJ) to repair DNA double-strand breaks (DSBs). Repair pathway choice is controlled by the activation and activity of pathways specific proteins in eukaryotes. Activity may be regulated by cell cycle stage, tissue type, and differentiation status. Bioflavonoids and other environmental agents such as pesticides have been shown to biochemically act as inhibitors of topoisomerase II (Top2). In cells, bioflavonoids directly lead to DNA double-strand breaks through both Top2-dependent and independent mechanisms, as well as induce DNA damage response (DDR) signaling, and promote alternative end-joining and chromosome alterations. This chapter will present differences in expression and activity of proteins in major DNA repair pathways, findings of Top2 inhibition by bioflavonoids and cellular response, discuss how these compounds trigger alternative end-joining, and conclude with implications for genome instability and human disease.

STimulator of INterferon Genes (STING) is an adaptor for cytoplasmic DNA sensing by cGAMP/cGAS that helps trigger innate immune responses (IIRs). Although STING is mostly localized in the ER, we find a separate inner nuclear membrane pool of STING that increases mobility and redistributes to the outer nuclear membrane upon IIR stimulation by transfected dsDNA or dsRNA mimic poly(I:C). Immunoprecipitation of STING from isolated nuclear envelopes coupled with mass spectrometry revealed a distinct nuclear envelope-STING proteome consisting of known nuclear membrane proteins and enriched in DNA- and RNA-binding proteins. Seventeen of these nuclear envelope STING partners are known to bind direct interactors of IRF3/7 transcription factors, and testing a subset of these revealed STING partners SYNCRIP, MEN1, DDX5, snRNP70, RPS27a, and AATF as novel modulators of dsDNA-triggered IIRs. Moreover, we find that SYNCRIP is a novel antagonist of the RNA virus, influenza A, potentially shedding light on reports of STING inhibition of RNA viruses.

Abstract Introduction: Gynecologic cancer (GYNC) accounts for 6.3% of all cancers and is the fourth leading cause of cancer death for women in the U.S. Autoimmune diseases (AD) are among the top ten leading causes of death among U.S. women age 65 and under. This high mortality rate among women with AD has been linked to cancer and other causes. Considering cancer and AD are the accumulative effect of genetics and lifestyle, and 90-95% of all cancers are linked to lifestyle and environmental factors, a study to investigate the association between patient-level predictors and GYNC among AD patients in the U.S. was imperative. Methods: 2007-2013 data from Florida State Inpatient samples of the Healthcare Cost and Utilization Project were used. The study population (n=836,717) was restricted to women who had any AD. The outcome variable was diagnosis of GYNC. 36 categorical transformed variables including race/ethnicity, age, insurance type, income level, GYN procedures, and comorbidities were analyzed as predictive variables (PV). Bootstrap Forest (JMP pro13) were was used to create a 10,000-decision tree (DT) model to identify independent predictors of GYNC. Multiple outputs such as confusion matrix, column contribution, and ROC was created for predictive analytics. Results: The analytical model used was confirmed by evaluating criteria such as AUC (0.82), sensitivity (0.68), specificity (0.82), false positive rate (0.18), wrongly predicted value (0.24), and overall accurate prediction (0.76) of the model. Using column contribution and single DT, the PV hysterectomy was the highest predictor of GYNC, followed by PV age;, and comorbidities such as diabetes, and obesity; along with race/ethnicity; and income level. Also, subpopulations of AD patients at risk for GYNC based on unique combinations of risk factors were established, including subpopulations such as women with AD, involved glandular disorders who had hysterectomy, and comorbidities such as diabetes and fluid and electrolyte disorders. Discussion: Both GYNC and AD are major chronic diseases among women. Because of high prevalence of AD among women, more elucidation on the association between GYNC and AD is essential for GYNC management. The unique combinations of characteristics that describe subgroups of patients at risk for GYNC among AD patients can be used as a potential risk assessment, as well as an early detection and/ or prevention tools for GYNC. Citation Format: Zahra Bahrani-Mostafavi, Larissa B. Huber, Wei Sha, Christine Richardson. Predictive analysis of gynecologic cancer risk factors using decision tree analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2341.

The abundance and diversity of intermediate filaments (IFs) in the C. elegans intestine indicate important contributions to intestinal function and organismal wellbeing. Fluorescent IF reporters localize below the actin-rich brush border and are highly enriched in the lumen-enveloping endotube, which is attached to the C. elegans apical junction. Mapping intestinal viscoelasticity by contact-free Brillouin microscopy reveals that the IF-rich endotube is positioned at the interface between the stiff brush border and soft cytoplasm suggesting a mechanical buffering function to deal with the frequent luminal distortions occurring during food intake and movement. In accordance, depletion of IFB-2, IFC-2 and IFD-2 leads to intestinal lumen dilation although depletion of IFC-1, IFD-1 and IFP-1 do not. Ultrastructural analyses of loss of function mutants further show that IFC-2 mutants have a rarefied endotube and IFB-2 mutants lack an endotube altogether. Remarkably, almost all IFB-2- and IFC-2-deficient animals develop to fertile adults. But developmental retardation, reduced brood size, altered survival and increased sensitivity to microbial toxin, osmotic and oxidative stress are seen in both mutants albeit to different degrees. Taken together, we propose that individual intestinal IF polypeptides contribute in different ways to endotube morphogenesis and cooperate to cope with changing environments.

Background: Etoposide is a well-characterized poison of topoisomerase II that promotes DNA double-strand breaks (DSBs) and is associated with therapy-related chromosomal translocation. Bioflavonoids have a similar chemical structure to etoposide, and some evidence from our lab and others suggests that they also induce DSBs and promote genome rearrangements. Since bioflavonoids are found in natural foods as well as in dietary supplements and energy drinks, quantification of their potency has important physiologic impact. The purpose of this study is to determine if subgroups of bioflavonoids induce DSBs by direct or indirect inhibition of topoisomerase II (topo II) and if the downstream damage signaling pathways and illegitimate repair are distinct. Methods: The experiments exposed a mouse embryonic stem cell Reporter cell line to physiologically relevant low and increasing doses of a panel of bioflavonoids. Following exposure, the kinetics of appearance of DNA damage was measured by immunocytochemistry and confocal microscopy to quantify γ-H2AX foci over time. Induction of DNA damage pathways was measured by Western blotting. Finally, appearance of chromosomal translocations was measured by appearance of GFP+ cells. To determine if bioflavonoid-induced DNA damage is directly through inhibition of topo II or an alternative mechanism, all experiments were performed in parallel in the presence of dexrazoxane, a topo II inhibitor. Results: Exposure to subgroups of bioflavonoids generated distinct amount of γ-H2AX foci. In addition, the kinetics of the appearance and repair of DNA damage differed between subgroups. Most bioflavonoid treatments showed persistent damage 8 hours post-exposure, though no treatment group caused as extensive damage as etoposide. Comparison of γ-H2AX foci immediately after bioflavonoid exposure with and without pretreatment with dexrazoxane demonstrated that bioflavonoids differ in their action in a topo II-dependent or -independent manner. Exposure to luteolin and myricetin after pretreatment with dexrazoxane produced a reduction in the presence of γ-H2AX foci. By contrast, exposure to genistein after pretreatment with dexrazoxane produced no change, and exposure to kaempferol or quercetin after pretreatment produced increased presence of γ-H2AX foci. Downstream induction of signaling pathways and formation of chromosomal translocations following exposure to bioflavonoids has been characterized. Current approaches are using pretreatment with dexrazoxane to also determine if signaling and repair patterns are distinct between the bioflavonoid subgroups. Conclusions: Bioflavonoids are known to induce pleiotropic effects on cells. These data suggest that the subgroup of bioflavonoid has a distinct mechanism of action to induce DNA damage in either a topo II-dependent or -independent manner, which may have implications for the potential of individual bioflavonoids to promote genome instability and chromosomal translocations. Citation Format: Donna A. Goodenow, Anindita Ghosh, Kiran Lalwani, Christine Richardson. Characterization of both topoisomerase II-dependent and -independent induction of DNA double-strand breaks, damage signaling pathways, and chromosomal translocations by subgroups of bioflavonoids [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A09.

DNA single-strand breaks (SSBs) represent the most abundant type of DNA damage. Unrepaired SSBs impair DNA replication and transcription, leading to cancer and neurodegenerative disorders. Although PARP1 and XRCC1 are implicated in the SSB repair pathway, it remains unclear how SSB repair and SSB signaling pathways are coordinated and regulated. Using Xenopus egg extract and in vitro reconstitution systems, here we show that SSBs are first sensed by APE1 to initiate 3'-5' SSB end resection, followed by APE2 recruitment to continue SSB end resection. Notably, APE1's exonuclease activity is critical for SSB repair and SSB signaling pathways. An APE1 exonuclease-deficient mutant identified in somatic tissue from a cancer patient highlighted the significance of APE1 exonuclease activity in cancer etiology. In addition, APE1 interacts with APE2 and PCNA, although PCNA is dispensable for APE1's exonuclease activity. Taken together, we propose a two-step APE1 /APE2-mediated mechanism for SSB end resection that couples DNA damage response with SSB repair in a eukaryotic system.

A double‐strand break (DSB) in one deoxyribonucleic acid (DNA) double helix, caused by DNA damaging agents such as ionising radiation or normal metabolic processes, can stimulate repair through multiple pathways. Homologous recombination (HR) utilises a second unbroken DNA double helix containing homologous sequences as a donor of genetic information to restore the intact DNA structure, while nonhomologous end joining (NHEJ) relegates broken DNA ends together with little to no homology. Deficiency in these repair pathways can cause genome instability can drive carcinogenesis. DSB stimulated repair can cause illegitimate HR between heterologous chromosome of the ligation of ends of DNA from heterologous chromosomes. Exogenous and endogenous agents can cause deoxyribonucleic acid (DNA) breaks leading to genome instability. Repair of double‐strand breaks (DSB) in eukaryotes occurs by either homology‐dependent or homology‐independent mechanisms. Homologous recombination (HR) facilitates high‐fidelity repair of DSBs and other plausible DNA damage. Site‐specific DSBs within the genome are used to study the explicit mechanism involved in the DSB repair. Unlike other model organisms, HR in higher eukaryotes requires additional protein factors. Mutations in HR proteins can increase predisposition to cancer.