Repair Of Double-strand DNA Breaks Research Articles

Genotoxicity associated with retroviral CAR transduction of ATM-deficient T cells.

Somatic variants in DNA damage-response genes such as ATM are widespread in hematologic malignancies. ATM protein is essential for double-strand DNA break repair. Germline ATM-deficiencies underlie ataxia-telangiectasia (A-T), a disease manifested by radio-sensitivity, immunodeficiency and predisposition to lymphoid malignancies. A-T patients diagnosed with malignancies have poor tolerance to chemotherapy or radiation. We investigated chimeric-antigen receptor (CAR) T cells using primary T-cells from patients with A-T (ATM-/-), heterozygote donors (ATM+/-) and healthy donors. ATM-/- T-cells proliferate and can be successfully transduced with CARs, though functional impairment of ATM-/- CAR-T cells was observed. Retroviral transduction of the CAR in ATM-/- T-cells resulted in high rates of chromosomal lesions at CAR insertion sites, as confirmed by next-generation long-read sequencing. This work suggests that ATM is essential to preserve genome integrity of CAR-T cells during retroviral manufacturing, and its lack poses a risk of chromosomal translocations and potential leukemogenicity.

Exploring the intersection of epigenetics, DNA repair, and immunology from studies of ICF syndrome, an inborn error of immunity.

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, a rare autosomal recessive disorder, manifests with hypoglobulinemia and chromosomal instability accompanied by DNA hypomethylation. Pathological variants in the DNMT3B, ZBTB24, CDCA7, or HELLS genes underlie its etiology. Activated lymphocytes from patients often display distinctive multiradial chromosomes fused via pericentromeric regions. Recent studies have provided deeper insights into how pathological variants in ICF-related proteins cause DNA hypomethylation and chromosome instability. However, the understanding of the molecular pathogenesis underlying immunodeficiency is still in its nascent stages. In the past half-decade, the roles of CDCA7, HELLS, and ZBTB24 in classical non-homologous end joining during double-strand DNA break repair and immunoglobulin class-switch recombination (CSR) have been unveiled. Nevertheless, given the decreased all classes of immunoglobulins in most patients, CSR deficiency alone cannot fully account for the immunodeficiency. The latest finding showing dysregulation of immunoglobulin signaling may provide a clue to understanding the immunodeficiency mechanism. While less common, a subgroup of patients exhibits T-cell abnormalities alongside B-cell anomalies, including reduced regulatory T-cells and increased effector memory T- and follicular helper T-cells. The dysregulation of immunoglobulin signaling in B-cells, the imbalance in T-cell subsets, and/or satellite RNA-mediated activation of innate immune response potentially explain autoimmune manifestations in a subset of patients. These findings emphasize the pivotal roles of ICF-related proteins in both B- and T-cell functions. ICF syndrome studies have illuminated many fundamental mechanisms. Further investigations will certainly continue to unveil additional mechanisms and their interplay.

Precise CRISPR/Cpf1 genome editing system in the Deinococcus radiodurans with superior DNA repair mechanisms

Deinococcus radiodurans, with its high homologous recombination (HR) efficiency of double-stranded DNA breaks (DSBs), is a model organism for studying genome stability maintenance and an attractive microbe for industrial applications. Here, we developed an efficient CRISPR/Cpf1 genome editing system in D. radiodurans by evaluating and optimizing double-plasmid strategies and four Cas effector proteins from various organisms, which can precisely introduce different types of template-dependent mutagenesis without off-target toxicity. Furthermore, the role of DNA repair genes in determining editing efficiency in D. radiodurans was evaluated by introducing the CRISPR/Cpf1 system into 13 mutant strains lacking various DNA damage response and repair factors. In addition to the crucial role of RecA-dependent HR required for CRISPR/Cpf1 editing, D. radiodurans showed higher editing efficiency when lacking DdrB, the single-stranded DNA annealing (SSA) protein involved in the RecA-independent DSB repair pathway. This suggests a possible competition between HR and SSA pathways in the CRISPR editing of D. radiodurans. Moreover, off-target effects were observed during the genome editing of the pprI knockout strain, a master DNA damage response gene in Deinococcus species, which suggested that precise regulation of DNA damage response is critical for a high-fidelity genome editing system.

Cryo-EM structures of RAD51 assembled on nucleosomes containing a DSB site.

RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs)1,2. However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.

Selective inhibition of DNA ligase IV provides additional efficacy to the treatment of anaplastic thyroid cancer.

Although the incidence of anaplastic thyroid carcinoma (ATC) is low (2.5% of thyroid cancer cases), this cancer has a very poor prognosis (survival rates < 5 months) and accounts for 14-39% of deaths. Conventional therapies based on surgery in combination with radiotherapy or chemotherapy showed limited effectiveness primarily due to the robust and protective DNA damage response in thyroid cancer cells. We used single-cell transcriptomic data from patients with different subtypes of thyroid cancer to study expression of genes involved in homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Then, we investigated the mechanisms of DNA damage and repair in anaplastic (C643 and Hth74) and papillary (TPC-1) thyroid cancer cell lines. The effect of caffeine (inhibitor of ATM and ATR) and UCN-01 (CHK1 inhibitor) was evaluated in cell cycle progression of thyroid cancer cells after γ-radiation or doxorubicin treatment. The DNA damage response was monitored after staining of phosphorylated γ-H2AX and 53BP1. Reporter plasmids were used to determine the efficacy of double-strand DNA breaks (DSBs) repair by HR and NHEJ in thyroid cancer cells. We evaluated the combination of selective inhibition of the DNA ligase IV by SCR7 and doxorubicin on cellular apoptosis and tumor growth in xenograft murine models of anaplastic thyroid cancer. Single-cell RNA-Seq showed that NHEJ- and HR-related genes are expressed in ATC and PTC patients. We showed that ATC cells undergo mitosis in the presence of unrepaired DNA damage caused by γ-radiation and doxorubicin treatment. To proliferate and survive, these cells efficiently repair DNA lesions using homologous recombination (HR) and non-homologous end joining (NHEJ). The combination of SCR7 with doxorubicin, significantly increased apoptosis and impaired ATC tumor growth in a xenograft mouse model compared to doxorubicin monotherapy. This study shows the therapeutic value of the combination of a DNA ligase IV inhibitor and DNA-damaging agents (doxorubicin and/or γ-radiation) for the treatment of anaplastic thyroid cancer.

Abstract B052: The role of LMO2 in DNA damage repair pathway choice in metastatic breast cancer

Abstract Introduction: Previously we discovered that a novel metastasis-initiating cell (MIC) population is marked by LMO2, a transcriptional adaptor protein in hematopoietic stem cells, and a T-cell oncogene. Knocking down LMO2 did not affect the growth of primary tumors, but did significantly reduce the number of circulating tumor cells and lung metastases. Transcriptional analysis of LMO2 knockdown shows the HALLMARK_DNA_REPAIR pathway is upregulated, suggesting that LMO2 is involved in regulating DNA repair in breast cancer cells. Interestingly, a previous study reported that in diffuse large B-cell lymphoma high LMO2 expression results in defective repair of double-stranded DNA breaks (Parvin et al., Cancer Cell, 36, 3 [2019]). We hypothesize that in breast cancer LMO2 expression is regulating the choice between homology-directed repair (HDR), an error-free method of DNA repair, and non-homologous end-joining (NHEJ), a more error-prone method. Cells that use NHEJ to repair DNA are dependent on poly (ADP-ribose) polymerase (PARP) and can be targeted with PARP inhibitors. LMO2 could thus be used as a new biomarker for benefit from PARP inhibition. Methods: LMO2 was identified using single-cell RNAseq data from human breast cancer patients. In vitro and in vivo assays using breast cancer cell lines and patient-derived xenografts were used to characterize the effect of LMO2 on metastasis. To study the role of LMO2 in the DNA damage response, we used MDA-MB-468 and HCC1806 cells with control and 2 independent shRNAs targeting LMO2. Immunofluorescent staining for γH2AX, 53BP1, and RAD51 was performed to measure changes in DNA damage and repair markers. Control and LMO2 knockdown cells were treated with olaparib, with cellular proliferation measured with a WST-1 assay and colony formation measured with crystal violet staining. Results and Conclusion: Knocking down LMO2 expression results in decreased NHEJ and increased HDR in MDA-MB-468 and HCC1806 cells. There is no significant difference in DNA damage overall as measured by γH2AX foci/nucleus. LMO2 knockdown in MDA-MB-468 cells reduces sensitivity to PARP inhibition with olaparib, as measured by an increase in the IC50 values. Our preliminary data suggest that LMO2 expression likely regulates the choice between NHEJ and HDR. LMO2 expression introduces a bias towards NHEJ, which sensitizes cells to PARP inhibition. This suggests that LMO2+ cells could be targeted with PARP inhibition, which could thus be used to target metastasis-initiating cells. Citation Format: Isobel Fetter, Veronica Haro-Acosta, Shaheen Sikandar. The role of LMO2 in DNA damage repair pathway choice in metastatic breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B052.

Structure of Escherichia coli exonuclease VII

Exonuclease VII (ExoVII) is a ubiquitous bacterial nuclease. Encoded by the xseA and xseB genes, ExoVII participates in multiple nucleic acid-dependent pathways including the processing of multicopy single-stranded DNA and the repair of covalent DNA-protein crosslinks (DPCs). Although many biochemical properties of ExoVII have been defined, little is known about its structure/function relationships. Here, we use cryoelectron microscopy (cryoEM) to determine that Escherichia coli ExoVII comprises a highly elongated XseA4·XseB24 holo-complex. Each XseA subunit dimerizes through a central extended α-helical segment decorated by six XseB subunits and a C-terminal, domain-swapped β-barrel element; two XseA2·XseB12 subcomplexes further associate using N-terminal OB (oligonucleotide/oligosaccharide-binding) folds and catalytic domains to form a spindle-shaped, catenated octaicosamer. The catalytic domains of XseA, which adopt a nuclease fold related to 3-dehydroquinate dehydratases, are sequestered in the center of the complex and accessible only through large pores formed between XseA tetramers. The architectural organization of ExoVII, combined with biochemical studies, indicate that substrate selectivity is controlled by steric access to its nuclease elements and that tetramer dissociation results from substrate DNA binding. Despite a lack of sequence and fold homology, the physical organization of ExoVII is reminiscent of Mre11·Rad50/SbcCD ATP (adenosine triphosphate)-dependent nucleases used in the repair of double-stranded DNA breaks, including those formed by DPCs through aberrant topoisomerase activity, suggesting that there may have been convergent evolutionary pressure to contend with such damage events.

KuINins as a New Class of HIV-1 Inhibitors That Block Post-Integration DNA Repair.

Integration of HIV-1 genomic cDNA results in the formation of single-strand breaks in cellular DNA, which must be repaired for efficient viral replication. Post-integration DNA repair mainly depends on the formation of the HIV-1 integrase complex with the Ku70 protein, which promotes DNA-PK assembly at sites of integration and its activation. Here, we have developed a first-class inhibitor of the integrase-Ku70 complex formation that inhibits HIV-1 replication in cell culture by acting at the stage of post-integration DNA repair. This inhibitor, named s17, does not affect the main cellular function of Ku70, namely its participation in the repair of double-strand DNA breaks through the non-homologous end-joining pathway. Using a molecular dynamics approach, we have constructed a model for the interaction of s17 with Ku70. According to this model, the interaction of two phenyl radicals of s17 with the L76 residue of Ku70 is important for this interaction. The requirement of two phenyl radicals in the structure of s17 for its inhibitory properties was confirmed using a set of s17 derivatives. We propose to stimulate compounds that inhibit post-integration repair by disrupting the integrase binding to Ku70 KuINins.

ATM/ATR Phosphorylation of CtIP on Its Conserved Sae2-like Domain Is Required for Genotoxin-Induced DNA Resection but Dispensable for Animal Development.

Homology-directed repair (HDR) of double-strand DNA breaks (DSBs) is dependent on enzymatic resection of DNA ends by the Mre11/Rad50/Nbs1 complex. DNA resection is triggered by the CtIP/Sae2 protein, which allosterically promotes Mre11-mediated endonuclease DNA cleavage at a position internal to the DSB. Although the mechanics of resection, including the initial endonucleolytic step, are largely conserved in eucaryotes, CtIP and its functional counterpart in Saccharomyces cerevisiae (Sae2) share only a modest stretch of amino acid homology. Nonetheless, this stretch contains two highly conserved phosphorylation sites for cyclin-dependent kinases (T843 in mouse) and the damage-induced ATM/ATR kinases (T855 in mouse), both of which are required for DNA resection. To explore the function of ATM/ATR phosphorylation at Ctip-T855, we generated and analyzed mice expressing the Ctip-T855A mutant. Surprisingly, unlike Ctip-null mice and Ctip-T843A-expressing mice, both of which undergo embryonic lethality, homozygous CtipT855A/T855A mice develop normally. Nonetheless, they are hypersensitive to ionizing radiation, and CtipT855A/T855A mouse embryo fibroblasts from these mice display marked defects in DNA resection, chromosomal stability, and HDR-mediated repair of DSBs. Thus, although ATM/ATR phosphorylation of CtIP-T855 is not required for normal animal development, it enhances CtIP-mediated DNA resection in response to acute stress, such as genotoxin exposure.

Abstract B065: Enhancing the therapeutic gain of proton radiotherapy in Locally Advanced Rectal Cancer (LARC) through manipulation of WSB1, ATM, and β-Catenin/c-Myc pathway Ubiquitination

Abstract Just 20% of patients with LARC attain a complete response to preoperative chemoradiotherapy which portends an excellent prognosis and opportunity to preserve the rectum through nonoperative surveillance. Moreover, normal tissue radiation toxicity can lead to significant morbidity, and becomes an even more important consideration in non-operative approaches where irradiated tissue is not resected. The geometry of conventional proton beams is already being used clinically to spare patient’s normal tissue, but the biology of entrance (ENT) vs. Bragg Peak (BP) protons has yet to be fully exploited to enhance tumor efficacy. Protons, unlike conventional x-ray radiation, spare normal tissue by lacking an exit dose - but they also exhibit a Bragg Peak (BP), which causes more complex DNA damage and is more biologically effective. ATM is critical in the repair of double strand DNA breaks (DSBs) and our lab has demonstrated that inhibitors of ATM (iATM) preferentially radiosensitize BP protons in CRC (Fig. 1A). Overactivation of the Wnt/β-catenin pathway via loss of function of APC is the most common genetic mutation in colorectal cancer (75-80%) and β-catenin over-expression negatively correlates with prognosis. WSB1 has been discovered by our lab to regulate ATM ubiquitination and degradation, it decreases metastates-free survival in CRC, and also forms a positive feedback loop with β-catenin and c-Myc that drives oncogenic progression. It is unknown if this upregulation of WSB1 promotes ATM degredation in LARC specifically, and a deubiquitinase (DUB) for ATM (which would counter WSB1) has yet to be identified. We preformed a comprehensive database search (UbiBrowser 2.0) for the most likely predicted DUBs for ATM (Fig. 1B), several of which have been identified as regulators of β-catenin/c-Myc, DSB repair pathways, and correlated with poor prognosis in CRC. We have also performed a screen of DUBs predicted to interact with ATM, and have preliminary evidence suggesting USP14 as the worlds first identified DUB of ATM (Fig 1C). USP14 expression is upregulated in CRC compared to surrounding normal tissue, promotes radioresistance, and promotes Wnt/β-catenin signalling. Higher USP14 expression in CRC is associated with liver and lymph node metastases and worse overall survival. USP14 regulates β-catenin by deubiquitinating K63-linked polyubiquitin chains of Dvl, and several small molecule inhibitors of USP14 have been identified. Our lab has preliminary data demonstrating USP14 is preferentially activated after BP proton irradiation (Fig. 1D), suggesting ATM may be necessary for repair of complex DSBs. While more work will be necessary to confirm these findings, many of the other predicted DUBs of ATM have overlapping functions regulating DSB repair and/or β-catenin/c-Myc signalling. We hypothesize that targeting ATM ubiquitination will open a therapeutic window in LARC by disrupting β-catenin/c-Myc signalling and selectively radiosensitizing BP protons. Citation Format: Sonja Dragojevic, Cameron M. Callaghan. Enhancing the therapeutic gain of proton radiotherapy in Locally Advanced Rectal Cancer (LARC) through manipulation of WSB1, ATM, and β-Catenin/c-Myc pathway Ubiquitination [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B065.

Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes

Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.

Reduction of double-strand DNA break repair exacerbates vascular aging.

Advanced age is the greatest risk factor for cardiovascular disease (CVD), the leading cause of death. Arterial function is impaired in advanced age which contributes to the development of CVD. One underexplored hypothesis is that DNA damage within arteries leads to this dysfunction, yet evidence demonstrating the incidence and physiological consequences of DNA damage in arteries, and in particular, in the microvasculature, in advanced age is limited. In the present study, we began by assessing the abundance of DNA damage in human and mouse lung microvascular endothelial cells and found that aging increases the percentage of cells with DNA damage. To explore the physiological consequences of increases in arterial DNA damage, we evaluated measures of endothelial function, microvascular and glycocalyx properties, and arterial stiffness in mice that were lacking or heterozygous for the double-strand DNA break repair protein ATM kinase. Surprisingly, in young mice, vascular function remained unchanged which led us to rationalize that perhaps aging is required to accumulate DNA damage. Indeed, in comparison to wild type littermate controls, mice heterozygous for ATM that were aged to ~18 mo (Old ATM +/-) displayed an accelerated vascular aging phenotype characterized by increases in arterial DNA damage, senescence signaling, and impairments in endothelium-dependent dilation due to elevated oxidative stress. Furthermore, old ATM +/- mice had reduced microvascular density and glycocalyx thickness as well as increased arterial stiffness. Collectively, these data demonstrate that DNA damage that accumulates in arteries in advanced age contributes to arterial dysfunction that is known to drive CVD.

Double-strand DNA break repair: molecular mechanisms and therapeutic targets.

Double-strand break (DSB), a significant DNA damage brought on by ionizing radiation, acts as an initiating signal in tumor radiotherapy, causing cancer cells death. The two primary pathways for DNA DSB repair in mammalian cells are nonhomologous end joining (NHEJ) and homologous recombination (HR), which cooperate and compete with one another to achieve effective repair. The DSB repair mechanism depends on numerous regulatory variables. DSB recognition and the recruitment of DNA repair components, for instance, depend on the MRE11-RAD50-NBS1 (MRN) complex and the Ku70/80 heterodimer/DNA-PKcs (DNA-PK) complex, whose control is crucial in determining the DSB repair pathway choice and efficiency of HR and NHEJ. In-depth elucidation on the DSB repair pathway's molecular mechanisms has greatly facilitated for creation of repair proteins or pathways-specific inhibitors to advance precise cancer therapy and boost the effectiveness of cancer radiotherapy. The architectures, roles, molecular processes, and inhibitors of significant target proteins in the DSB repair pathways are reviewed in this article. The strategy and application in cancer therapy are also discussed based on the advancement of inhibitors targeted DSB damage response and repair proteins.

Characterization of DNA Damage Response-Associated Somatic Mutations in Borderline Resectable and Locally Advanced Pancreatic Cancer

The role of radiation for pancreatic cancer remains controversial, with recent studies showing conflicting results, highlighting the need to develop biomarkers of radiation response. Despite its potential utility in predicting radiosensitivity, the landscape of somatic mutations in borderline resectable pancreatic cancer (BRPC) and locally advanced pancreatic cancer (LAPC), as related to DNA damage response (DDR), has not been well characterized. This study aimed to characterize the frequency of such mutations in a cohort of patients with BRPC/LAPC treated with neoadjuvant chemotherapy and stereotactic body radiotherapy (SBRT). Mutational data was collected from patients with BRPC/LAPC treated at a single institution with neoadjuvant chemotherapy and SBRT, followed by surgical resection from 2016-2021. Chemotherapy consisted of modified FOLFIRINOX or gemcitabine/nab-paclitaxel, and patients were treated with SBRT in 33 Gy in 5 fractions. Genomic data was obtained from either endoscopic biopsy or surgical specimens, and next-generation sequencing was performed either in-house with a Solid Tumor Panel or with FoundationOne CDx. Specific emphasis was placed on the characterization of double-strand DNA break (DSB) repair genes, as this is the type of tumor cell damage traditionally induced by radiation therapy. Genes associated with the two main pathways of DSB repair, non-homologous end joining (NHEJ) and homologous repair (HR), were analyzed. Specific HR pathway mutations assessed were BLM, BRCA1/2, MRE11, NBN, PALB2, RAD50, RAD51B-D, and RAD54L, while PRKDC mutations were assessed for the NHEJ pathway. Mutations in ATM, an important initiator of DDR pathways, were also analyzed. Additionally, the frequency of mutations in TP53, CDKN2A and SMAD4 in patients with concomitant KRAS mutations was assessed. Eighty-five patients were included in the study. Five (5.9%) patients had mutations in the NHEJ pathway of the PRKDC gene. Twenty (23.5%) patients had mutations in the HR pathway, including BRCA2 (10/85; 11.8%), PALB2 (5/85; 5.9%), BRCA1 (3/85; 3.5%), and RAD50 (1/85; 1.2%). Six (7.1%) patients had mutations in ATM. No patients were found to have mutations in BLM, RAD51B-D, RAD54L, or NBN. Amongst patients with KRAS mutations (72/85), concomitant mutations were observed in TP53 (47/85; 55.3%), CDKN2A (16/85; 18.8%), and SMAD4 (9/85; 10.6%). Herein, we characterized the frequency of somatic mutations associated with DSB repair genes in patients with BRPC/LAPC. Data analysis on outcomes related to radiation response in patients with mutations in DDR pathways is ongoing, but will likely also benefit from multi-institutional efforts to increase the power to answer this question.

The molecular details of a novel phosphorylation-dependent interaction between MRN and the SOSS complex.

The repair of double-strand DNA breaks (DSBs) by homologous recombination is crucial in the maintenance of genome integrity. While the key role of the Mre11-Rad50-Nbs1 (MRN) complex in repair is well known, hSSB1 (SOSSB and OBFC2B), one of the main components of the sensor of single-stranded DNA (SOSS) protein complex, has also been shown to rapidly localize to DSB breaks and promote repair. We have previously demonstrated that hSSB1 binds directly to Nbs1, a component of the MRN complex, in a DNA damage-independent manner. However, recruitment of the MRN complex has also been demonstrated by an interaction between Integrator Complex Subunit 3 (INTS3; also known as SOSSA), another member of the SOSS complex, and Nbs1. In this study, we utilize a combined approach of in silico, biochemical, and functional experiments to uncover the molecular details of INTS3 binding to Nbs1. We demonstrate that the forkhead-associated domain of Nbs1 interacts with INTS3 via phosphorylation-dependent binding to INTS3 at Threonine 592, with contributions from Serine 590. Based on these data, we propose a model of MRN recruitment to a DSB via INTS3.

Protein Phosphatase 4 Is Required for Centrobin Function in DNA Damage Repair.

Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein phosphatase 4 (PP4) plays a pivotal role in coordinating cellular response to DNA damage. Meanwhile, Centrobin (CNTRB), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with CNTRB in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and CNTRB and shed light on the importance of two specific motifs in CNTRB, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells depleted of PP4 or CNTRB and cells harboring FRVP and SQ mutations in CNTRB, which result in similar abnormal chromosome morphologies. This phenomenon likely results from the impaired resolution of Holliday junctions, which serve as crucial intermediates in HR. Taken together, our results provide new insights into the intricate mechanisms of PP4 and CNTRB-regulated HR repair and their interrelation.

Parp Inhibitors and Radiotherapy: A New Combination for Prostate Cancer (Systematic Review).

PARPi, in combination with ionizing radiation, has demonstrated the ability to enhance cellular radiosensitivity in different tumors. The rationale is that the exposure to radiation leads to both physical and biochemical damage to DNA, prompting cells to initiate three primary mechanisms for DNA repair. Two double-stranded DNA breaks (DSB) repair pathways: (1) non-homologous end-joining (NHEJ) and (2) homologous recombination (HR); and (3) a single-stranded DNA break (SSB) repair pathway (base excision repair, BER). In this scenario, PARPi can serve as radiosensitizers by leveraging the BER pathway. This mechanism heightens the likelihood of replication forks collapsing, consequently leading to the formation of persistent DSBs. Together, the combination of PARPi and radiotherapy is a potent oncological strategy. This combination has proven its efficacy in different tumors. However, in prostate cancer, there are only preclinical studies to support it and, recently, an ongoing clinical trial. The objective of this paper is to perform a review of the current evidence regarding the use of PARPi and radiotherapy (RT) in PCa and to give future insight on this topic.

Rapid Genotyping of Nothobranchius furzeri Embryos, Larvae, and Adult Fish via High-Resolution Melt Analysis (HRMA).

CRISPR-Cas9 has eased the induction of sequence-specific mutations and has therefore become a powerful tool to generate mutant lines for studying the role of specific genes. The cellular repair of Cas9-induced double-stranded DNA breaks by the error-prone nonhomologous end-joining pathway can result in various indel mutations. Having identified and chosen a specific mutation in a target gene, the establishment of a respective mutant line requires a feasible and precise method to differentiate the genotypes of the offspring. Here, we provide a protocol that allows genotyping of large numbers of Nothobranchius furzeri embryos, larvae, and adults harboring a previously identified indel or point mutation in a short time via high-resolution melt analysis (HRMA).

Phosphoregulation of the checkpoint kinase Mec1ATR

Yeast Mec1, and its mammalian ortholog, Ataxia-Telangiectasia and Rad3-related, are giant protein kinases central to replication stress and double strand DNA break repair. Mec1ATR, in complex with Ddc2ATRIP, is a ‘sensor’ of single stranded DNA, and phosphorylates numerous cell cycle and DNA repair factors to enforce cell cycle arrest and facilitate repair. Over the last several years, new techniques — particularly in structural biology — have provided molecular mechanisms for Mec1ATR function. It is becoming increasingly clear how post-translational modification of Mec1ATR and its interaction partners modulates the DNA damage checkpoint. In this review, we summarise the most recent work unravelling Mec1ATR function in the DNA damage checkpoint and provide a molecular context for its regulation by phosphorylation.

O-075 Deficiency of DNA double-strand break repair in human preimplantation embryos revealed by CRISPR-Cas9

Abstract Study question Is double-strand DNA break repair functional in human preimplantation embryos and what implications might a deficiency have for genome editing performed at early developmental stages? Summary answer CRISPR-Cas9 succeeds in generating DNA breaks with high efficiency, but these often remain unresolved, indicating that early human embryos are deficient in DNA repair. What is known already Genome editing (GE) technologies, such as CRISPR-Cas9, raise the possibility of avoiding transmission of inherited disorders by converting mutant genes back to wild-type (normal). The therapeutic application of CRISPR-Cas9 at the preimplantation stage is controversial due to uncertainty over safety and concerns about the alteration of the human germline. However, from a technical perspective it remains highly attractive, since it is the only developmental stage where delivery of CRISPR-Cas9 to every cell of the individual can be guaranteed. Currently, information on safety aspects and efficacy are lacking. Therefore, evaluation of the cellular response to CRISPR-Cas9 in human embryos is needed. Study design, size, duration 84 embryos were generated for research in an IRB approved study. For this purpose, donor oocytes were fertilised with donor sperm using ICSI. 51 of the resulting embryos served as controls, while in the other 33 double strand DNA breaks (DSBs) were created in a highly controlled fashion, directed at specific genomic sites using CRISPR-Cas9 technology. Successfully fertilised oocytes underwent culture in a time-lapse incubator and the duration of key developmental events were carefully timed. Participants/materials, setting, methods CRISPR-Cas9 components targeted several non-coding sites in the genome. An artificial DNA fragment was provided, which cells could potentially use for homology directed repair (HDR). Embryos were grown for 60 hours, then disaggregated. Blastomeres underwent whole genome amplification, followed by next generation sequencing to detect segmental aneuploidy related to failure of DNA repair. Additionally, sites targeted by CRISPR-Cas9 were amplified and sequenced to confirm whether repair had occurred and to distinguish the specific mechanism employed. Main results and the role of chance The study successfully validated and utilised three CRISPR ‘guides’ that targeted sites on chromosomes 2, 3 and 12. Alterations at the targeted sites were detected in 24/25 embryos (96% targeting efficiency). Repair of DSBs created by CRISPR-Cas9 was considered successful when an embryo contained at least one blastomere with changes to the DNA sequence at the targeted site consistent with nonhomologous end joining (NHEJ) or HDR. In total, 53 double-stranded breaks were generated by CRISPR-Cas9 during this study. Of these, 51% (27) were repaired by NHEJ, 9% (5) repaired by HDR and 40% (21) remained unresolved, the persistent breakage eventually causing segmental aneuploidy with a breakpoint at the targeted side. Segmental abnormalities are known to be detrimental to embryonic viability and risk congenital abnormalities in offspring. This study shows that human embryos, prior to activation of the genome, have a DNA damage repair deficiency. Timings of morphokinetic milestones were altered in embryos with unresolved DNA damage, confirming that checkpoint control is active. However, continued mitotic division, despite the presence of DSBs, shows cellular mechanisms that usually preserve genetic integrity lack stringency, ultimately failing to ensure repair. The results provide a warning against the therapeutic use of CRISPR-Cas9 in human embryos. Limitations, reasons for caution While targeting the embryonic genome using CRISPR-Cas9 was remarkably efficient (DSBs introduced in 96% of embryos), the majority of cells utilised NHEJ for repair, a process allowing introduction of additional mutations rather than correcting existing ones. Only &amp;lt;10% of cells used the HDR repair mechanism necessary for removal of mutations. Wider implications of the findings These findings reveal that cells of early human embryos are less able to process DSBs than other cell types. This suggests that embryo culture methods should give consideration to protecting the genome from damage. The results caution against clinical application of genome editing technologies that create DSBs, such as CRISPR-Cas9. Trial registration number NA