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Abstract Background Inherited variations in DNA double-strand break (DSB) repair pathway are known to influence ovarian cancer occurrence, progression and treatment response. Despite its significance, survival-associated genetic variants within the DSB pathway remain underexplored. Methods In the present study, we performed a two-phase analysis of 19,290 single-nucleotide polymorphisms (SNPs) in 199 genes in the DSB repair pathway from a genome-wide association study (GWAS) dataset and explored their associations with overall survival (OS) in 1039 Han Chinese epithelial ovarian carcinoma (EOC) patients. After utilizing multivariate Cox regression analysis with bayesian false-discovery probability for multiple test correction, significant genetic variations were identified and subsequently underwent functional prediction and validation. Results We discovered a significant association between poor overall survival and the functional variant GEN1 rs56070363 C > T (CT + TT vs. TT, adjusted hazard ratio (HR) = 2.50, P T on survival was attributed to its reduced binding affinity to hsa-miR-1287-5p and the resultant upregulation of GEN1 mRNA expression. Overexpression of GEN1 aggregated EOC cell proliferation, invasion and migration presumably by influencing the expression of immune inhibitory factors, thereby elevating the proportion of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and then constructing an immunosuppressive tumor microenvironment. Conclusions In conclusion, GEN1 rs56070363 variant could serve as a potential predictive biomarker and chemotherapeutic target for improving the survival of EOC patients.

Background: Inherited variations in DNA double-strand break (DSB) repair pathway are known to influence ovarian cancer occurrence, progression and treatment response. Despite its significance, survival-associated genetic variants within the DSB pathway remain underexplored. Methods: In the present study, we performed a two-phase analysis of 19,290 single-nucleotide polymorphisms (SNPs) in 199 genes in the DSB repair pathway from a genome-wide association study (GWAS) dataset and explored their associations with overall survival (OS) in 1039 Han Chinese epithelial ovarian carcinoma (EOC) patients. After utilizing multivariate Cox regression analysis with bayesian false-discovery probability for multiple test correction, significant genetic variations were identified and subsequently underwent functional prediction and validation. Results: We discovered a significant association between poor overall survival and the functional variant GEN1 rs56070363 C > T (CT + TT vs. TT, adjusted hazard ratio (HR) = 2.50, P < 0.001). And the impact of GEN1 rs56070363 C > T on survival was attributed to its reduced binding affinity to hsa-miR-1287-5p and the resultant upregulation of GEN1 mRNA expression. Overexpression of GEN1 aggregated EOC cell proliferation, invasion and migration presumably by influencing the expression of immune inhibitory factors, thereby elevating the proportion of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and then constructing an immunosuppressive tumor microenvironment. Conclusions: In conclusion, GEN1 rs56070363 variant could serve as a potential predictive biomarker and chemotherapeutic target for improving the survival of EOC patients. Highlights: Inherited variations in DSB repair pathway are known to influence ovarian cancer occurrence, progression and treatment response. In the present study, we performed a two-phase analysis of 19,290 SNPs in 199 genes in the DSB repair pathway among 1,039 ovarian cancer patients. Ultimately, we identified that the poor overall survival was significantly associated with functional variant GEN1 rs56070363 C > T. Further investigations revealed the mechanism underlying this association: the C > T transition resulted in decreased binding affinity to hsa-miR-1287-5p and subsequent upregulation of GEN1 mRNA expression. Overexpression of GEN1 aggregated EOC cell proliferation, invasion and migration presumably by influencing the expression immune inhibitory factors, thereby elevating the proportion of PMN-MDSCs and then constructing an immunosuppressive tumor microenvironment. Our findings thus provide novel molecular targets and a theoretical basis for individualized treatment approaches in ovarian cancer. [ABSTRACT FROM AUTHOR]

Chrysin, a natural compound isolated from various plants, such as the blue passion flower (Passiflora caerulea L.), exhibits multiple pharmacological activities, such as antitumor, anti-inflammatory and antioxidant activities. Accumulating evidence shows that chrysin inhibits cancer cell growth by inducing apoptosis and regulating cell cycle arrest. However, whether chrysin is involved in regulating genomic stability and its underlying mechanisms in breast cancer cells have not been determined. Here, we demonstrated that chrysin impairs genomic stability in MCF-7 and BT474 cells, inhibits cell survival and enhances the sensitivity of MCF-7 cells to chemotherapeutic drugs. Further experiments revealed that chrysin impairs DNA double-strand break (DSB) repair, resulting in accumulation of DNA damage. Mechanistic studies showed that chrysin inhibits the recruitment of the key NHEJ factor 53BP1 and delays the recruitment of the HR factor RAD51. Thus, we elucidated novel regulatory mechanisms of chrysin in DSB repair and proposed that a combination of chrysin and chemotherapy has curative potential in breast cancers. [ABSTRACT FROM AUTHOR]

Abstract Background The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated. Methods and results Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls. Conclusions Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.

The fine tuning of the DNA double strand break repair pathway choice relies on different regulatory layers that respond to environmental and local cues. Among them, the presence of non-canonical nucleic acids structures seems to create challenges for the repair of nearby DNA double strand breaks. In this review, we focus on the recently published effects of G-quadruplexes and R-loops on DNA end resection and homologous recombination. Finally, we hypothesized a connection between those two atypical DNA structures in inhibiting the DNA end resection step of HR. [ABSTRACT FROM AUTHOR]

The fine tuning of the DNA double strand break repair pathway choice relies on different regulatory layers that respond to environmental and local cues. Among them, the presence of non-canonical nucleic acids structures seems to create challenges for the repair of nearby DNA double strand breaks. In this review, we focus on the recently published effects of G-quadruplexes and R-loops on DNA end resection and homologous recombination. Finally, we hypothesized a connection between those two atypical DNA structures in inhibiting the DNA end resection step of HR.

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.

Background: The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated. Methods and results: Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls. Conclusions: Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease. [ABSTRACT FROM AUTHOR]

Mutations caused by DNA damage are a main driver of cancer. We discovered that recognition of newly synthesised histone H4 directs breast cancer type 1 susceptibility protein (BRCA1) to post-replicative chromatin. The switch from mutagenic to error-free DNA double strand break repair by homologous recombination is therefore controlled by chromatin.

Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes. [ABSTRACT FROM AUTHOR]

Homologous recombination (HR) is an evolutionarily conserved process that plays a pivotal role in the equilibrium between genetic stability and diversity. HR is commonly considered to be error-free, but several studies have shown that HR can be error-prone. Here, we discuss the actual accuracy of HR.First, we present the product of genetic exchanges (gene conversion, GC, and crossing over, CO) and the mechanisms of HR during double strand break repair and replication restart. We discuss the intrinsic capacities of HR to generate genome rearrangements by GC or CO, either during DSB repair or replication restart. During this process, abortive HR intermediates generate genetic instability and cell toxicity. In addition to genome rearrangements, HR also primes error-prone DNA synthesis and favors mutagenesis on single stranded DNA, a key DNA intermediate during the HR process. The fact that cells have developed several mechanisms protecting against HR excess emphasize its potential risks. Consistent with this duality, several pro-oncogenic situations have been consistently associated with either decreased or increased HR levels. Nevertheless, this versatility also has advantages that we outline here.We conclude that HR is a double-edged sword, which on one hand controls the equilibrium between genome stability and diversity but, on the other hand, can jeopardize the maintenance of genomic integrity. Therefore, whether non-homologous end joining (which, in contrast with HR, is not intrinsically mutagenic) or HR is the more mutagenic process is a question that should be re-evaluated. Both processes can be Dr. Jekyll in maintaining genome stability/variability and Mr. Hyde in jeopardizing genome integrity.

Glioblastoma is deemed the most malignant form of brain tumour, particularly due to its resistance to conventional treatments. A small surviving group of aberrant stem cells termed glioma initiation cells (GICs) that escape surgical debulking are suggested to be the cause of this resistance. Relatively quiescent in nature, GICs are capable of driving tumour recurrence and undergo lineage differentiation. Most importantly, these GICs are resistant to radiotherapy, suggesting that radioresistance contribute to their survival. In a previous study, we demonstrated that GICs had a restricted double strand break (DSB) repair pathway involving predominantly homologous recombination (HR) associated with a lack of functional G 1 /S checkpoint arrest. This unusual behaviour led to less efficient non-homologous end joining (NHEJ) repair and overall slower DNA DSB repair kinetics. To determine whether specific targeting of the HR pathway with small molecule inhibitors could increase GIC radiosensitivity, we used the Ataxia-telangiectasia mutated inhibitor (ATMi) to ablate HR and the DNA-dependent protein kinase inhibitor (DNA-PKi) to inhibit NHEJ. Pre-treatment with ATMi prior to ionizing radiation (IR) exposure prevented HR-mediated DNA DSB repair as measured by Rad51 foci accumulation. Increased cell death in vitro and improved in vivo animal survival could be observed with combined ATMi and IR treatment. Conversely, DNA-PKi treatment had minimal impact on GICs ability to resolve DNA DSB after IR with only partial reduction in cell survival, confirming the major role of HR. These results provide a mechanistic insight into the predominant form of DNA DSB repair in GICs, which when targeted may be a potential translational approach to increase patient survival. [ABSTRACT FROM AUTHOR]

Homologous recombination (HR) is an evolutionarily conserved process that plays a pivotal role in the equilibrium between genetic stability and diversity. HR is commonly considered to be error-free, but several studies have shown that HR can be error-prone. Here, we discuss the actual accuracy of HR. First, we present the product of genetic exchanges (gene conversion, GC, and crossing over, CO) and the mechanisms of HR during double strand break repair and replication restart. We discuss the intrinsic capacities of HR to generate genome rearrangements by GC or CO, either during DSB repair or replication restart. During this process, abortive HR intermediates generate genetic instability and cell toxicity. In addition to genome rearrangements, HR also primes error-prone DNA synthesis and favors mutagenesis on single stranded DNA, a key DNA intermediate during the HR process. The fact that cells have developed several mechanisms protecting against HR excess emphasize its potential risks. Consistent with this duality, several pro-oncogenic situations have been consistently associated with either decreased or increased HR levels. Nevertheless, this versatility also has advantages that we outline here. We conclude that HR is a double-edged sword, which on one hand controls the equilibrium between genome stability and diversity but, on the other hand, can jeopardize the maintenance of genomic integrity. Therefore, whether non-homologous end joining (which, in contrast with HR, is not intrinsically mutagenic) or HR is the more mutagenic process is a question that should be re-evaluated. Both processes can be ''Dr. Jekyll'' in maintaining genome stability/variability and ''Mr. Hyde'' in jeopardizing genome integrity. [ABSTRACT FROM AUTHOR]

Tc1, one of the founding members of the Tc1/ mariner transposon superfamily, was identified in the nematode Caenorhabditis elegans more than 25 years ago. Over the years, Tc1 and other endogenous mariner transposons became valuable tools for mutagenesis and targeted gene inactivation in C. elegans. However, transposition is naturally repressed in the C. elegans germline by an RNAi-like mechanism, necessitating the use of mutant strains in which transposition was globally derepressed, which causes drawbacks such as uncontrolled proliferation of the transposons in the genome and accumulation of background mutations. The more recent mobilization of the Drosophila mariner transposon Mos1 in the C. elegans germline circumvented the problems inherent to endogenous transposons. Mos1 transposition strictly depends on the expression of the Mos transposase, which can be controlled in the germline using inducible promoters. First, Mos1 can be used for insertional mutagenesis. The mobilization of Mos1 copies present on an extrachromosomal array results in the generation of a small number of Mos1 genomic insertions that can be rapidly cloned by inverse PCR. Second, Mos1 insertions can be used for genome engineering. Triggering the excision of a genomic Mos1 insertion causes a chromosomal break, which can be repaired by transgene-instructed gene conversion. This process is used to introduce specific changes in a given gene, such as point mutations, deletions or insertions of a tag, and to create single-copy transgenes. [ABSTRACT FROM AUTHOR]

Abstract: Benzene causes hematotoxicity and leukemia in humans. To analyze benzene-caused aberrant gene expression, we examined differential gene expression by microarray analysis of peripheral mononuclear blood cells from seven workers diagnosed with benzene poisoning and seven matched controls. Twenty-two genes were found up-regulated and 18 down-regulated in benzene patients compared with controls. Here we report the characterization of two benzene-regulated genes. CYP4F3A, which encodes the leukotriene B4 (LTB4) ω-hydroxylase, is important for inactivation of LTB4 in neutrophils. CYP4F3A mRNA was found elevated in all patients; moreover, CYP4F3A mRNA and protein were induced by benzene metabolite phenol in HL-60 and K562 cells as well as ex vivo in human peripheral neutrophils. Silencing of CYP4F3A in HL-60 cells by lentiviral delivery of CYP4F3A-specific siRNA reduced cell survival to 56%, 44%, 22%, 14%, and 3% at 3, 4, 5, 6, and 7 days, respectively; the results suggest that CYP4F3A is a critical positive regulator of HL-60 proliferation. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates non-homologous end joining (NHEJ) in DNA double strand break (DSB) repair. DNA-PKcs mRNA was found consistently increased in the patients and DNA-PKcs mRNA and protein were induced by hydroquinone in HL-60 cells. In a DSB model, hydroquinone induced the formation of γ-H2AX foci, a marker of DSBs, in HL-60 cells. The findings indicate that hydroquinone induces DSBs and induction correlates with elevated levels of DNA-PKcs and NHEJ. Similar results were obtained in K562 cells treated with phenol. Since NHEJ is error-prone, induction of DNA-PKcs and NHEJ may contribute to mutagenesis and leukemia by benzene. To our knowledge, the study demonstrated for the first time that benzene and metabolites induce CYP4F3A and DNA-PKcs both in vivo and in vitro. Induction of the genes may play a role in the pathogenesis of benzene hematotoxicity and serve as biomarkers of benzene exposure. [Copyright &y& Elsevier]

We examined radiosensitizing properties of two novel platinum complexes (ethylenediamine(l-malato)platinum(II)), Pt1 and bis(1-ethylimidazole(l-malato)platinum(II)), Pt4. Initial double strand break (DSB) level and DSB rejoining were measured, using pulse field gel electrophoresis (PFGE) in human G1 phase lymphocytes subjected to Pt complex treatment alone and in combination with 10 Gy of X-rays. Effects of Pt complex pre-treatment followed by X-irradiation were examined on survival (clonogenic ability) and growth (48 h growth tests) in Chinese hamster ovary (CHO-K1), xrs6 and L5178Y (LY) cells (LY-R and LY-S sublines). Cell cycle distributions of CHO cells after drug treatment were determined with the use of flow cytometry.Pt1 slowed down rejoining of X-ray induced DSB. It exerted a more than additive lethal effect on CHO-K1 cells but not on L5178Y cells subjected to combined Pt complex treatment and X-irradiation. In xrs6 cells the effect of combined Pt1+X treatment was additive. We conclude that, as earlier proposed for other Pt complexes, the radiosensitizing effect of Pt1 is connected with converting repairable DNA damage into irrepairable one (mode (i) of action). The requirements for this mode of sensitization are functional DNA repair systems (nucleotide excision repair (NER) and non-homologous end-joining (NHEJ)).Pt4 does not slow down DSB rejoining. It shows a considerable ability to arrest cells in G2 phase. We assume that Pt4 pre-treatment arrests cells in G2 phase and thus sensitizes to X-rays these cells that have a radiosensitive G2 phase (mode (ii) of action). [Copyright &y& Elsevier]

Protein arginine methyltransferase 1 (PRMT1) is a major enzyme responsible for the formation of methylarginine in mammalian cells; however, its function in vivo is not well understood due to its early embryonic lethality in null mice exhibiting spontaneous DNA damage, cell cycle delays, and defects in check point activation. Here, we generated germ cell-specific Prmt1 knock-out (KO) mice to evaluate the function of PRMT1 in spermatogenesis. Our findings demonstrate that PRMT1 is vital for male fertility in mice. Spermatogenesis in Prmt1 KO mice was arrested at the zygotene-like stage of the first meiotic division due to an elevated number of DNA double-strand breaks (DSBs). There was a loss of methylation in meiotic recombination 11 (MRE11), the key endonuclease in MRE11/RAD50/NBS 1 (MRN) complex, resulting in the accumulation of SPO11 protein in DSBs. The ATM-mediated negative feedback control over SPO11 was lost and, consequently, the repair pathway of DSBs was highly affected in PRMT1 deficient male germ cells. Our findings provide a novel insight into the role of PRMT1-mediated asymmetric demethylation in mouse spermatogenesis. [ABSTRACT FROM AUTHOR]

Simple Summary: Colorectal cancer (CRC) is among the most common cancers and the third leading cause of cancer deaths worldwide. Despite the identification of alterations in DNA repair genes and the resulting genomic instability in sub-populations of CRC, therapies that exploit defects in DNA repair pathways or high level of replicative stress have been explored only in breast, ovarian, and other tumor types, but not yet systematically in CRC. Here, we discuss how targeting genes involved in the responses to replication stress and the repair of DNA double-strand breaks (DSBs) could provide new therapeutic opportunities to treat CRCs and have the potential to confer increased sensitivity to current chemotherapy regimens, thus, expanding the spectrum of therapy options, and potentially improving clinical outcomes for CRC patients. Despite the ample improvements of CRC molecular landscape, the therapeutic options still rely on conventional chemotherapy-based regimens for early disease, and few targeted agents are recommended for clinical use in the metastatic setting. Moreover, the impact of cytotoxic, targeted agents, and immunotherapy combinations in the metastatic scenario is not fully satisfactory, especially the outcomes for patients who develop resistance to these treatments need to be improved. Here, we examine the opportunity to consider therapeutic agents targeting DNA repair and DNA replication stress response as strategies to exploit genetic or functional defects in the DNA damage response (DDR) pathways through synthetic lethal mechanisms, still not explored in CRC. These include the multiple actors involved in the repair of DNA double-strand breaks (DSBs) through homologous recombination (HR), classical non-homologous end joining (NHEJ), and microhomology-mediated end-joining (MMEJ), inhibitors of the base excision repair (BER) protein poly (ADP-ribose) polymerase (PARP), as well as inhibitors of the DNA damage kinases ataxia-telangiectasia and Rad3 related (ATR), CHK1, WEE1, and ataxia-telangiectasia mutated (ATM). We also review the biomarkers that guide the use of these agents, and current clinical trials with targeted DDR therapies. [ABSTRACT FROM AUTHOR]

The BRCA1-BARD1 complex directs the DNA double-strand break (DSB) repair pathway choice to error-free homologous recombination (HR) during the S-G2 stages. Targeting BRCA1-BARD1 to DSB-proximal sites requires BARD1-mediated nucleosome interaction and histone mark recognition. Here, we report the cryo-EM structure of BARD1 bound to a ubiquitinated nucleosome core particle (NCPUb) at 3.1 Å resolution and illustrate how BARD1 simultaneously recognizes the DNA damage-induced mark H2AK15ub and DNA replication-associated mark H4K20me0 on the nucleosome. In vitro and in vivo analyses reveal that the BARD1-NCPUb complex is stabilized by BARD1-nucleosome interaction, BARD1-ubiquitin interaction, and BARD1 ARD domain-BARD1 BRCT domain interaction, and abrogating these interactions is detrimental to HR activity. We further identify multiple disease-causing BARD1 mutations that disrupt BARD1-NCPUb interactions and hence impair HR. Together, this study elucidates the mechanism of BRCA1-BARD1 complex recruitment and retention by DSB-flanking nucleosomes and sheds important light on cancer therapeutic avenues. [Display omitted] • Cryo-EM structure of human BARD1 in complex with H2AK15-ubiquitinated nucleosome • The BARD1 BUDR motif contacts the nucleosome acidic patch and ubiquitin K63-E64 • The BARD1 ARD domain grips the H4 tail by accommodating the unmethylated H4K20 • Mutations disrupting BARD1-NCPUb association impair homologous recombination The recruitment of BRCA1-BARD1 complex to DSB sites requires BARD1-mediated dual histone mark recognition, thereby promoting HR in S/G2 cells. Dai et al. determined the structure of BARD1-NCPH2AK15Ub complex, showing how BARD1 reads dual histone marks in the nucleosomal context to direct BRCA1 recruitment. [ABSTRACT FROM AUTHOR]

Simple Summary: The combination of RT and the first generation AR blockers to improve the outcome in prostate cancer remain a matter of controversial debate in clinical trials. In the current study we aim to investigate the effect of three FDA approved second-generation antiandrogens (abiraterone acetate, apalutamide and enzalutamide), as more potent inhibitors of the AR signaling, on the cytotoxicity of RT in pre-clinical models. In vitro and ex vivo analyses revealed a strong radiosensitising effect for the second-generation antiandrogens, regardless of the castration state. The first-generation AR-blocker bicalutamide failed to show any radiosensitising effect. The radiosensitising effect of the second-generation antiandrogens was attributed to the inhibition of DSB repair. Together, we provide a proof-of-principle pre-clinical evidence to rationalize the clinical use of the second-generation antiandrogens to enhance the effect of IR as a potential strategy to improve the outcomes of PCa patients with localized disease who undergo ablative RT. (1) Background: The combination of the first-generation antiandrogens and radiotherapy (RT) has been studied extensively in the clinical setting of prostate cancer (PCa). Here, we evaluated the potential radiosensitizing effect of the second-generation antiandrogens abiraterone acetate, apalutamide and enzalutamide. (2) Methods: Cell proliferation and agarose-colony forming assay were used to measure the effect on survival. Double strand break repair efficiency was monitored using immunofluorescence staining of γH2AX/53BP1. (3) Results: We report retrospectively a minor benefit for PCa patients received first-generation androgen blockers and RT compared to patients treated with RT alone. Combining either of the second-generation antiandrogens and 2Gy suppressed cell growth and increased doubling time significantly more than 2Gy alone, in both hormone-responsive LNCaP and castration-resistant C4-2B cells. These findings were recapitulated in resistant sub-clones to (i) hormone ablation (LNCaP-abl), (ii) abiraterone acetate (LNCaP-abi), (iii) apalutamide (LNCaP-ARN509), (iv) enzalutamide (C4-2B-ENZA), and in castration-resistant 22-RV1 cells. This radiosensitization effect was not observable using the first-generation antiandrogen bicalutamide. Inhibition of DNA DSB repair was found to contribute to the radiosensitization effect of second-generation antiandrogens, as demonstrated by a significant increase in residual γH2AX and 53BP1 foci numbers at 24h post-IR. DSB repair inhibition was further demonstrated in 22 patient-derived tumor slice cultures treated with abiraterone acetate before ex-vivo irradiation with 2Gy. (4) Conclusion: Together, these data show that second-generation antiandrogens can enhance radiosensitivity in PCa through DSB repair inhibition, regardless of their hormonal status. Translated into clinical practice, our results may help to find additional strategies to improve the effectiveness of RT in localized PCa, paving the way for a clinical trial. [ABSTRACT FROM AUTHOR]

Reduced NME1 expression in melanoma cell lines, mouse models of melanoma, and melanoma specimens in human patients is associated with increased metastatic activity. Herein, we investigate the role of NME1 in repair of double-stranded breaks (DSBs) and choice of double-strand break repair (DSBR) pathways in melanoma cells. Using chromatin immunoprecipitation, NME1 was shown to be recruited rapidly and directly to DSBs generated by the homing endonuclease I-PpoI. NME1 was recruited to DSBs within 30 min, in concert with recruitment of ataxia-telangiectasia mutated (ATM) protein, an early step in DSBR complex formation, as well as loss of histone 2B. NME1 was detected up to 5 kb from the break site after DSB induction, suggesting a role in extending chromatin reorganization away from the repair site. shRNA-mediated silencing of NME1 expression led to increases in the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways of double-strand break repair (DSBR), and reduction in the low fidelity, alternative-NHEJ (A-NHEJ) pathway. These findings suggest low expression of NME1 drives DSBR towards higher fidelity pathways, conferring enhanced genomic stability necessary for rapid and error-free proliferation in invasive and metastatic cells. The novel mechanism highlighted in the current study appears likely to impact metastatic potential and therapy-resistance in advanced melanoma and other cancers. [ABSTRACT FROM AUTHOR]

Lung cancer has the highest incidence and mortality among all cancers, with non-small cell lung cancer (NSCLC) accounting for 85-90% of all lung cancers. Here we investigated the function of COMMD1 in the repair of DNA double strand breaks (DSBs) and as a prognostic and therapeutic target in NSCLC. COMMD1 function in DSB repair was investigated using reporter assays in COMMD1-siRNA-depleted cells. The role of COMMD1 in NSCLC was investigated using bioinformatic analysis, qRT-PCR and immunoblotting of control and NSCLC cells, tissue microarrays, cell viability and cell cycle experiments. DNA repair assays demonstrated that COMMD1 is required for the efficient repair of DSBs and reporter assays showed that COMMD1 functions in both non-homologous-end-joining and homologous recombination. Bioinformatic analysis showed that COMMD1 is upregulated in NSCLC, with high levels of COMMD1 associated with poor patient prognosis. COMMD1 mRNA and protein were upregulated across a panel of NSCLC cell lines and siRNA-mediated depletion of COMMD1 decreased cell proliferation and reduced cell viability of NSCLC, with enhanced death after exposure to DNA damaging-agents. Bioinformatic analyses demonstrated that COMMD1 levels positively correlate with the gene ontology DNA repair gene set enrichment signature in NSCLC. Taken together, COMMD1 functions in DSB repair, is a prognostic maker in NSCLC and is potentially a novel anti-cancer therapeutic target for NSCLC.

Mutations caused by DNA damage are a main driver of cancer. We discovered that recognition of newly synthesised histone H4 directs breast cancer type 1 susceptibility protein (BRCA1) to post-replicative chromatin. The switch from mutagenic to error-free DNA double strand break repair by homologous recombination is therefore controlled by chromatin. [ABSTRACT FROM AUTHOR]

Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes.