Maternal gene Ooep may participate in homologous recombination-mediated DNA double-strand break repair in mouse oocytes

DNA damage in oocytes can cause infertility and birth defects. DNA double-strand breaks （DSBs） are highly deleterious and can substantially impair genome integrity. Homologous recombination （HR）-mediated DNA DSB repair plays dominant roles in safeguarding oocyte quantity and quality. However, little is known regarding the key players of the HR repair pathway in oocytes. Here, we identified oocyte-specific gene Ooep as a novel key component of the HR repair pathway in mouse oocytes. OOEP was required for efficient ataxia telangiectasia mutated （ATM） kinase activation and Rad51 recombinase （RAD51） focal accumulation at DNA DSBs. Ooep null oocytes were defective in DNA DSB repair and prone to apoptosis upon exogenous DNA damage insults. Moreover, Ooep null oocytes exhibited delayed meiotic maturation. Therefore, OOEP played roles in preserving oocyte quantity and quality by maintaining genome stability. Ooep expression decreased with the advance of maternal age, suggesting its involvement in maternal aging.

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.

Target binding and residence:a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is widely used for targeted genomic and epigenomic modifications and imaging in cells and organisms,and holds tremendous promise in clinical applications.The efficiency and accuracy of the technology are partly determined by the target binding affinity and residence time of Cas9-single-guide RNA(sgRNA)at a given site.However,little attention has been paid to the effect of target binding affinity and residence duration on the repair of Cas9-induced DNA double-strand breaks(DSBs).We propose that the choice of DSB repair pathway may be altered by variation in the binding affinity and residence duration of Cas9-sgRNA at the cleaved target,contributing to significantly heterogeneous mutations in CRISPR/Cas9 genome editing.Here,we discuss the effect of Cas9-sgRNA target binding and residence on the choice of DSB repair pathway in CRISPR/Cas9 genome editing,and the opportunity this presents to optimize Cas9-based technology.

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.

Role of deubiquitinating enzymes in DNA double-strand break repair

DNA is the hereditary material in humans and almost all other organisms. It is essential for maintaining accurate transmission of genetic information. In the life cycle, DNA replication, cell division, or genome damage, including that caused by endogenous and exogenous agents, may cause DNA aberrations. Of all forms of DNA damage, DNA double-strand breaks(DSBs) are the most serious. If the repair function is defective, DNA damage may cause gene mutation, genome instability, and cell chromosome loss, which in turn can even lead to tumorigenesis. DNA damage can be repaired through multiple mechanisms. Homologous recombination(HR) and non-homologous end joining(NHEJ) are the two main repair mechanisms for DNA DSBs. Increasing amounts of evidence reveal that protein modifications play an essential role in DNA damage repair.Protein deubiquitination is a vital post-translational modification which removes ubiquitin molecules or polyubiquitinated chains from substrates in order to reverse the ubiquitination reaction. This review discusses the role of deubiquitinating enzymes(DUBs) in repairing DNA DSBs. Exploring the molecular mechanisms of DUB regulation in DSB repair will provide new insights to combat human diseases and develop novel therapeutic approaches.

p53 and its isoforms in DNA double-stranded break repair

DNA double-stranded break(DSB)is one of the most catastrophic damages of genotoxic insult.Inappropriate repair of DNA DSBs results in the loss of genetic information,mutation,and the generation of harmful genomic rearrangements,which predisposes an organism to immunodeficiency,neurological damage,and cancer.The tumor repressor p53 plays a key role in DNA damage response,and has been found to be mutated in 50%of human cancer.p53,p63,and p73 are three members of the p53 gene family.Recent discoveries have shown that human p53 gene encodes at least 12 isoforms.Different p53 members and isoforms play various roles in orchestrating DNA damage response to maintain genomic integrity.This review briefly explores the functions of p53 and its isoforms in DNA DSB repair.

Exploiting DNA repair pathways for tumor sensitization,mitigation of resistance,and normal tissue protection in radiotherapy

More than half of cancer patients are treated with radiotherapy,which kills tumor cells by directly and indirectly inducing DNA damage,including cytotoxic DNA double-strand breaks(DSBs).Tumor cells respond to these threats by activating a complex signaling network termed the DNA damage response(DDR).The DDR arrests the cell cycle,upregulates DNA repair,and triggers apoptosis when damage is excessive.The DDR signaling and DNA repair pathways are fertile terrain for therapeutic intervention.This review highlights strategies to improve therapeutic gain by targeting DDR and DNA repair pathways to radiosensitize tumor cells,overcome intrinsic and acquired tumor radioresistance,and protect normal tissue.Many biological and environmental factors determine tumor and normal cell responses to ionizing radiation and genotoxic chemotherapeutics.These include cell type and cell cycle phase distribution;tissue/tumor microenvironment and oxygen levels;DNA damage load and quality;DNA repair capacity;and susceptibility to apoptosis or other active or passive cell death pathways.We provide an overview of radiobiological parameters associated with X-ray,proton,and carbon ion radiotherapy;DNA repair and DNA damage signaling pathways;and other factors that regulate tumor and normal cell responses to radiation.We then focus on recent studies exploiting DSB repair pathways to enhance radiotherapy therapeutic gain.

CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

With its high efficiency for site-specific genome editing and easy manipulation,the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated protein 9(CAS9)system has become the most widely used gene editing technology in biomedical research.In addition,significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases,several of which are entering clinical trials.Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA(gRNA)in human cells,promoting genomic DNA double-stranded break(DSB)damage and genomic instability.CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase(DNA-PK)complex and disrupts the interaction between KU86 and its kinase subunit,leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining(NHEJ)pathway.XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility,and dCAS9 is a CAS9 variant without nuclease activity.We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair.Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival,our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application.

The DNA damage and regulatory strategy in hematopoietic stem cells after irradiation exposure:Progress and challenges

The hematopoietic system is susceptible to ionizing radiation(IR),which can cause acute hematopoietic failure or long-term myelosuppression.As the most primitive cells of the hematopoietic hierarchy,hematopoietic stem cells(HSCs)maintain lifelong hematopoietic homeostasis and promote hematopoietic regeneration during stress.Numerous studies have shown that nuclear and mitochondrial genomes are the main targets of radiation injury in HSCs.More importantly,the damage of DNA may trigger a series of biological responses that largely determine HSC fate following IR exposure.Although some essential pathways and factors involved in DNA injury and damage in HSCs have been revealed,a comprehensive understanding of the biological effects of radiation on HSCs still needs to be improved.This review focuses on recent insights into the molecular mechanisms underlying DNA damage and repair in HSCs after IR.Then summarize corresponding regulatory measures,which may provide a reference for further research in this field.

Profiling of the assembly of RecA nucleofilaments implies a potential target for environmental factors to disturb DNA repair

Double-strand breaks(DSBs),one class of the most harmful DNA damage forms that bring elevated health risks,need to be repaired timely and effectively.However,an increasing number of environmental pollutants have been identified to impair DSB repair from various mechanisms.Our previous work indicated that the formation of unsaturated Rec A nucleofilaments plays an essential role in homology recombination(HR) pathway which can accurately repair DSBs.In this study,by developing a benzonase cutting protection assay and combining it with traditional electrophoretic mobility shift assay(EMSA) analysis,we further investigated the assembly patterns of four Rec A mutants that display differential DSB repair ability and ATPase activity.We observed that the mutants(G204S and S69G) possessing both ATP hydrolysis and DSB repair activities form unsaturated nucleofilaments similar to that formed by the wild type Rec A,whereas the other two ATP hydrolysis-deficient mutants(K72R and E96D) that fail to mediate HR form more compacted nucleofilaments in the presence of ATP.These results establish a coupling of ATPase activity and effective DSB repair ability via the assembly status of Rec A nucleofilaments.This linkage provides a potential target for environmental factors to disturb the essential HR pathway for DSB repair by suppressing the ATPase activity and altering the assembly pattern of nucleofilaments.

Genotypic characteristics of resistant tumors to pre-operative ionizing radiation in rectal cancer

Due to a wide range of clinical response in patients un-dergoing neo-adjuvant chemoradiation for rectal cancer it is essential to understand molecular factors that lead to the broad response observed in patients receiving the same form of treatment.Despite extensive research in this field,the exact mechanisms still remain elusive.Data raging from DNA-repair to specific molecules lead-ing to cell survival as well as resistance to apoptosis have been investigated.Individually,or in combination,there is no single pathway that has become clinically applicable to date.In the following review,we describe the current status of various pathways that might lead to resistance to the therapeutic applications of ionizing radiation in rectal cancer.