Unrepaired Double-strand Breaks Research Articles

Base excision repair and double strand break repair cooperate to modulate the formation of unrepaired double strand breaks in mouse brain

We lack the fundamental information needed to understand how DNA damage in the brain is generated and how it is controlled over a lifetime in the absence of replication check points. To address these questions, here, we integrate cell-type and region-specific features of DNA repair activity in the normal brain. The brain has the same repair proteins as other tissues, but normal, canonical repair activity is unequal and is characterized by high base excision repair (BER) and low double strand break repair (DSBR). The natural imbalance creates conditions where single strand breaks (SSBs) can convert to double strand breaks (DSBs) and reversibly switch between states in response to oxidation both in vivo and in vitro. Our data suggest that, in a normal background of repair, SSBs and DSBs are in an equilibrium which is pushed or pulled by metabolic state. Interconversion of SSB to DSBs provides a physiological check point, which would allow the formation of unrepaired DSBs for productive functions, but would also restrict them from exceeding tolerable limits.

Abstract Or106: DNA Double-stranded Breaks, a Hallmark of Aging, Defined at the Nucleotide Resolution, Are Increased and Associated with Transcription in the Cardiac Myocyte Genome in LMNA-Cardiomyopathy

Background: An intrinsic feature of gene transcription is the formation of DNA superhelices near the transcription bubble, which are resolved upon induction of transient double-stranded breaks (DSBs) by topoisomerases. Unrepaired DSBs are pathogenic as they lead to cell cycle arrest, senescence, inflammation, and organ dysfunction. Hypothesis and Aims: We posit that DSBs would be more prevalent at the genomic sites associated with gene expression. As Lamin-A (LMNA) regulates active transcription through Lamin associated domains (LADS) we predict for DSBs to be more common in non-LAD regions. The objectives were to identify and characterize genome-wide DSBs at the nucleotide resolution and determine the association of DSBs with transcription in cardiac myocytes. Methods: We identified the genome-wide DSBs in ~ 1 million cardiac myocytes per heart in 3 wild-type and 3 myocyte-specific LMNA-deficient ( Myh6-Cre:Lmna F/F ) mice by END-Sequencing, the LADs were defined in cardiac myocytes via LMNA CUT&RUN assay (N=5). Results: The prevalence of DSBs was 0.8% and 2.2% in the wild-type and Myh6-Cre:Lmna F/F myocytes, respectively. The END-Seq signals for 8 DSBs were enriched in the wild-type and 6,764 in Myh6-Cre:Lmna F/F myocytes, respectively (q<0.05). The DSBs were preferentially localized to the gene regions, transcription initiation sites, cardiac transcription factor motifs, and the G quadruplex forming structures. Because LMNA regulates transcription through the LADs, we defined the LADs in cardiac myocytes and found an average of 818 LADs per myocyte. LADs that were shared by at least three mice (N= 2,572) were considered constitutive LADs (cLADs), which comprised about a third of the mouse cardiac myocyte genomes. Transcript levels of the protein-coding genes located at the cLADs (N=3,975) were ~ 16-fold lower than those at the non-LAD regions (N=~17,778). The prevalence of DSBs was higher in the non-LAD as compared to the cLAD regions. Likewise, DSBs were more common in the loss-of LAD regions, defined as the juxtaposed genomic regions in the Myh6-Cre:Lmna F/F that corresponded to the LAD regions in the wild-type myocytes. Conclusions: To our knowledge, this is the first identification of DSBs, at the nucleotide resolution in the cardiovascular system. The prevalence of DSBs was higher in the genomic regions associated with transcription. Because transcription is pervasive, DSBs are expected to be common and pathogenic in various disease states and aging.

DNA double-stranded breaks, a hallmark of aging, defined at the nucleotide resolution, are increased and associated with transcription in the cardiac myocytes in LMNA-cardiomyopathy.

An intrinsic feature of gene transcription is the formation of DNA superhelices near the transcription bubble, which are resolved upon induction of transient double-stranded breaks (DSBs) by topoisomerases. Unrepaired DSBs are pathogenic as they lead to cell cycle arrest, senescence, inflammation, and organ dysfunction. We posit that DSBs would be more prevalent at the genomic sites that are associated with gene expression. The objectives were to identify and characterize genome-wide DSBs at the nucleotide resolution and determine the association of DSBs with transcription in cardiac myocytes. We identified the genome-wide DSBs in ∼1 million cardiac myocytes per heart in three wild-type and three myocyte-specific LMNA-deficient (Myh6-Cre:LmnaF/F) mice by END-Sequencing. The prevalence of DSBs was 0.8% and 2.2% in the wild-type and Myh6-Cre:LmnaF/F myocytes, respectively. The END-Seq signals were enriched for 8 and 6764 DSBs in the wild-type and Myh6-Cre:LmnaF/F myocytes, respectively (q < 0.05). The DSBs were preferentially localized to the gene regions, transcription initiation sites, cardiac transcription factor motifs, and the G quadruplex forming structures. Because LMNA regulates transcription through the lamin-associated domains (LADs), we defined the LADs in cardiac myocytes by a Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assay (N = 5). On average there were 818 LADs per myocyte. Constitutive LADs (cLADs), defined as LADs that were shared by at least three genomes (N = 2572), comprised about a third of the mouse cardiac myocyte genomes. Transcript levels of the protein-coding genes located at the cLADs (N = 3975) were ∼16-fold lower than those at the non-LAD regions (N = ∼17 778). The prevalence of DSBs was higher in the non-LAD as compared to the cLAD regions. Likewise, DSBs were more common in the loss-of-LAD regions, defined as the genomic regions in the Myh6-Cre:LmnaF/F that were juxtaposed to the LAD regions in the wild-type myocytes. To our knowledge, this is the first identification of the DSBs, at the nucleotide resolution in the cardiovascular system. The prevalence of DSBs was higher in the genomic regions associated with transcription. Because transcription is pervasive, DSBs are expected to be common and pathogenic in various states and aging.

Abstract PR013: Association of a polymorphic variant in JMJD1C with response to chemoradiotherapy in rectal cancer and head and neck cancer: Implications for a diverse population

Abstract Neoadjuvant chemoradiotherapy (nCRT) is the standard treatment for locally advanced rectal cancer (LARC). Although nCRT has improved outcomes for many patients, ~70-80% of patients show poor or no response, and CRT is toxic. No biomarker exists that can predict which patients may benefit from nCRT. CRT efficacy arises from unrepaired double-strand breaks (DSBs). Recent work has suggested that dynamic changes in lysine modification of chromatin regulate DSB repair. This study investigated the association of germline single nucleotide polymorphisms (SNPs) in lysine-modifying genes with DSB repair and CRT outcomes in LARC. The LARC patients (n=84) were segregated into poor (PoR) and complete (CR) responders using the Neoadjuvant Rectal score. Blood DNA was sequenced to identified SNPs, matched LARC patient-derived cells were tested for sensitivity to DNA damage, and results correlated. Significant SNP-response correlations were confirmed in an independent cohort of CRT-treated head and neck squamous cell carcinoma (HNSCC) patients who had received chemoradiation (n=90). The HNSCC patients were segregated into 3 groups based on response: disease-free (n=30), recurrent disease (n=28), and never-disease free (n=32). Isogenic HNSCC lines with either the wildtype (WT) or SNP were generated using CRISPR/Cas9 technology, and bulk RNA sequencing and immunofluorescence-based studies were performed to assess pathways affected. LARC CRs were significantly enriched versus PoRs for a germline SNP in the lysine H3K9 demethylase, JMJD1C, resulting in a missense variant (discovery (n=30, p&amp;lt;0.00001), validation (n=54, p=0.001); Fisher’s Exact). In the HNSCC cohort, the JMJD1C SNP was significantly associated with no recurrence (p=0.0003, Fisher’s Exact). LARC patient-derived cell lines carrying the SNP were significantly more sensitive to DSB damage versus those carrying the WT (n=12, p&amp;lt;0.001, Mann-Whitney). Cells bearing the SNP were significantly enriched for altered mRNA levels of genes associated with DNA repair compared with WT cells (q-value&amp;lt;0.05). On irradiation, the SNP cells showed robust activation of DSB repair (γH2AX, BRCA1 foci, JMJD1C nuclear localization) versus the WT cells. Finally, analysis of the JMJD1C SNP frequency in large population datasets found that while it was abundant in European Americans (EAs), it is relatively rare in African Americans (AAs) (EA= 0.3139 vs. AA=0.072; gnomAD population). This study provides novel insight into a SNP in a lysine-modifying gene affecting DSB repair that may serve as a predictive biomarker for CRT response in 2 different cancer types. Our data suggest that host genetics impact DSB repair and warrant further investigation to better understand disparities in response to CRT. Future research will further explore the racial/ethnic disparities in tumor recurrence and confirm results in in vitro and in vivo models of cancer, and in large patient datasets. Citation Format: Adria Hasan, Elena Demidova, Philip Czyzewicz, Shreya Shah, Karthik Devarajan, Thomas J. Galloway, Margret B. Einarson, Barbara Burtness, Erica A. Golemis, Joshua E. Meyer, Sanjeevani Arora. Association of a polymorphic variant in JMJD1C with response to chemoradiotherapy in rectal cancer and head and neck cancer: Implications for a diverse population [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr PR013.

Abstract PO-032: Association of a polymorphic variant in JMJD1C with tumor recurrence after adjuvant chemoradiation therapy in head and neck squamous cell carcinoma

Abstract Background: Adjuvant chemoradiation therapy (CRT) is the standard treatment for locally advanced head and neck squamous cell carcinomas (HNSCCs). Over 50% of HNSCC cases are diagnosed at the locally advanced stage, with in-field persistence and recurrence major causes of morbidity and poor survival outcome. Apart from human papillomavirus (HPV) status, there are no predictive biomarkers for CRT outcomes. CRT lethality arises from unrepaired double strand breaks (DSBs) in DNA. Recent work has suggested that dynamic changes in lysine modification of chromatin regulate DSB repair. This study investigated the association of germline single nucleotide polymorphisms (SNPs) in lysine-modifying genes with DSB repair and CRT outcomes in HNSCC. Methods: Blood specimens and associated retrospective clinical data from HNSCC patients (n=90; age range=41-89, median age 64; males=69, females=21) were obtained under IRB# 21-9921. The patients had HPV-negative tumors in the larynx or oral cavity, and had received radiation, with some receiving concurrent chemotherapy. Patients receiving concurrent chemotherapy were: 40% in disease-free group, 21% in recurrence disease group, and 38% in never disease-free group. The patients were segregated into 3 groups: disease-free (n=30), recurrent disease (n=28), and never-disease free (n=32). DNA from blood was genotyped for specific germline SNPs in lysine-modifying genes. Isogenic HNSCC lines (Cal-27, SCC9) carrying either the wild type (WT) or homozygous SNP were generated using CRISPR/Cas9 technology, and bulk RNA sequencing and immunofluorescence (IF)-based studies were performed to assess cellular pathways affected. Results: A homozygous variant SNP in the coding region of JMJD1C was significantly enriched in the disease-free group (n=21/30) versus the recurrent disease (n=3/28) and never disease-free (n=2/32) groups (p=0.0003, OR=0, Fisher’s Exact test). Analysis of SNP frequency in large population datasets found that while it was abundant in European Americans (EAs), it is relatively rare in African Americans (AAs) (EA= 0.3139 vs. AA=0.072; gnomAD population). Cal-27 JMJD1C SNP cells were significantly enriched for altered mRNA levels of genes associated with DNA repair, replication, cell cycle, oxidative phosphorylation, and histone kinase activity versus CAL-27 with WT JMJD1C (q-value&amp;lt;0.05). The SCC9 JMJD1C SNP cells were significantly enriched in extracellular matrix organization, cytokine-cytokine receptor interaction, and humoral immune response versus SCC9 with WT JMJD1C (q-value&amp;lt;0.05). On irradiation, the JMJD1C SNP cells had robust activation of the DSB repair pathway (BRCA1- mediated repair foci) versus the WT cell lines. Conclusion and future work: This work provides novel insight into a germline SNP in a lysine-modifying gene effecting DSB repair and associating with CRT outcomes in HNSCC. It may serve as a predictive biomarker for recurrence. Future research will explore racial disparities in tumor recurrence and confirm results in larger HNSCC datasets. Citation Format: Adria Hasan, Elena V. Demidova, Shreya M. Shah, Philip Czyzewicz, Karthik Devarajan, Thomas Galloway, Barbara Burtness, Erica A. Golemis, Joshua E. Meyer, Sanjeevani Arwesd. Association of a polymorphic variant in JMJD1C with tumor recurrence after adjuvant chemoradiation therapy in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-032.

Copy number alteration features in pan-cancer homologous recombination deficiency prediction and biology

Homologous recombination deficiency (HRD) renders cancer cells vulnerable to unrepaired double-strand breaks and is an important therapeutic target as exemplified by the clinical efficacy of poly ADP-ribose polymerase (PARP) inhibitors as well as the platinum chemotherapy drugs applied to HRD patients. However, it remains a challenge to predict HRD status precisely and economically. Copy number alteration (CNA), as a pervasive trait of human cancers, can be extracted from a variety of data sources, including whole genome sequencing (WGS), SNP array, and panel sequencing, and thus can be easily applied clinically. Here we systematically evaluate the predictive performance of various CNA features and signatures in HRD prediction and build a gradient boosting machine model (HRDCNA) for pan-cancer HRD prediction based on these CNA features. CNA features BP10MB[1] (The number of breakpoints per 10MB of DNA is 1) and SS[ > 7 & <=8] (The log10-based size of segments is greater than 7 and less than or equal to 8) are identified as the most important features in HRD prediction. HRDCNA suggests the biallelic inactivation of BRCA1, BRCA2, PALB2, RAD51C, RAD51D, and BARD1 as the major genetic basis for human HRD, and may also be applied to effectively validate the pathogenicity of BRCA1/2 variants of uncertain significance (VUS). Together, this study provides a robust tool for cost-effective HRD prediction and also demonstrates the applicability of CNA features and signatures in cancer precision medicine.

High-throughput measurement of double strand break global repair phenotype in peripheral blood mononuclear cells after long-term cryopreservation.

Peripheral blood mononuclear cells (PBMCs) are a useful model for biochemical assays, particularly for etiological studies. We describe here a method for measuring DNA repair capacity (DRC) in archival cryogenically preserved PBMCs. To model DRC, we measured γ-H2AX repair kinetics in thawed PBMCs after irradiation with 3Gy gamma rays. Time-dependent fluorescently labeled γ-H2AX levels were measured at five time points from 1 to 20 h, yielding an estimate of global DRC repair kinetics as well as a measure of unrepaired double strand breaks at 20 h. While γ-H2AX levels are traditionally measured by either microscopy or flow-cytometry, we developed a protocol for imaging flow cytometry (IFC) that combines the detailed information of microscopy with the statistical power of flow methods. The visual imaging component of the IFC allows for monitoring aspects such as cellular health and apoptosis as well as fluorescence localization of the γ-H2AX signal, which ensures the power and significance of this technique. Application of a machine-learning based image classification improved flow cytometry fluorescent measurements by identifying apoptotic cells unable to undergo DNA repair. We present here DRC repair parameters from 18 frozen archival PBMCs and 28 fresh blood samples collected from a demographically diverse cohort of women measured in a high-throughput IFC format. This thaw method and assay can be used alone or in conjunction with other assays to measure etiological phenotypes in cryogenic biobanks of PBMCs.

Medaka factor in the germline alpha (figα) is indispensable for oogenesis but not for gonadal sex differentiation

A basic helix-loop-helix transcription factor, figα, is one of the earliest marker genes of oocyte differentiation in vertebrates. In the present study, we made figα knockout medaka by CRISPR/Cas9, expecting aborted progress of oogenesis, to see if differentiation of somatic ovarian tissues is affected. Figα knockout male gonads differentiated normally into testes with functional sperm. The females, on the other hand, were sterile; there are oocytes only up to pachytene. No growing oocytes in diplotene were found. The phenotype was already apparent at 10 days after hatching, when diplotene oocytes start to develop in the control. Furthermore, several putative target genes of figα were not expressed in the mutant female gonads. Previous studies showed that medaka lacking germ cells have morphologically abnormal gonads and female to male sex reversal occurred. Figα knockout female gonads differentiated morphologically into ovaries, suggesting that in medaka, figα knockout prevents oogenesis progress into diplotene stage, and ovarian differentiation do not need developed oocytes beyond pachytene stage. This ovarian phenotype reminded us of "pachytene checkpoint", which blocks meiotic progress into diplotene when chromosomal abnormalities are present. Therefore, we further analyzed phenotype of oocytes in the mutant ovaries. The number of oocytes at each meiotic stage suggested that oogenesis normally proceeded up to pachytene stage. The number of apoptotic oocytes in the knockout ovaries was small. Finally, we did not detect any abnormal pairings or unrepaired double-strand breaks by immunostaining. Taken together, figα knockout likely stop oocyte growth at or around pachytene via unknown mechanisms other than "pachytene checkpoint" and arrested oocytes remained in the ovary. This is in contrast to figα knockout mice where all oocytes underwent apoptosis quickly after birth.

BMAL1 collaborates with CLOCK to directly promote DNA double-strand break repair and tumor chemoresistance

Accumulating evidence indicates a correlation between circadian dysfunction and genomic instability. However, whether the circadian machinery directly regulates DNA damage repair, especially in double-strand breaks (DSBs), remains poorly understood. Here, we report that in response to DSBs, BMAL1 is activated by ATM-mediated phosphorylation at S183. Phosphorylated BMAL1 is then localized to DNA damage sites, where it facilitates acetylase CLOCK to load in the chromatin, regulating the acetylation of histone H4 (H4Ac) at DSB sites. In this way, the BMAL1-CLOCK-H4Ac axis promotes the DNA end-resection to generate single-stranded DNA (ssDNA) and the subsequent homologous recombination (HR). BMAL1 deficient cells display defective HR, accumulation of unrepaired DSBs and genome instability. Accordingly, depletion of BMAL1 significantly enhances the sensitivity of adrenocortical carcinoma (ACC) to DNA damage-based therapy in vitro and in vivo. These findings uncover non-canonical function of BMAL1 and CLOCK in HR-mediated DSB repair, which may have an implication in cancer therapeutics.

Differential Expression of Nuclear Body Component, Zc3h8, Disrupts DNA Double Strand Break Repair

The processes of DNA damage recognition and repair are paramount to maintaining genome integrity in the cell. Failure to do so results in genetic instability and the accumulation of mutations. Double strand breaks have the greatest potential to introduce mutations because rearrangements, non‐homologous end joining, deletions, or insertions may result. Unrepaired or incorrectly repaired DNA double strand breaks can lead to cell death, apoptosis, or disease such as cancer. We previously demonstrated that the C3H1 zinc finger‐containing protein Zc3h8 (also called Fliz1) promotes oncogenic phenotypes when overexpressed in cells, while oncogenic phenotypes are suppressed when expression is reduced. In mice, Zc3h8 expression affected tumorigenesis and tumor size in vivo. Higher expression of Zc3h8 led to more frequent tumors and larger tumors in mice. In humans Zc3h8 is overexpressed in 2‐6% of cancers and is a statistically significant prognostic indicator for poor rates of survival. The Zc3h8 protein has been localized to nuclear bodies including promyelocytic leukemia (PML) and Cajal Bodies where key cellular processes including transcription, translation, post‐translational modifications, and RNA processing are regulated. PML Bodies, in particular, have a role in DNA damage response and repair since P53 is located in PML Bodies along with DAXX, ATRX, and CK2. Since Zc3h8 co‐localized with DNA damage response and repair proteins in PML bodies and promotes oncogenic properties in cells, we investigated if Zc3h8 expression alters how cells respond to DNA damage such as double strand breaks and how this damage is mitigated in cells. Differential expression of Zc3h8 affected mouse mammary tumor cell’s ability to repair double strand breaks caused by the topoisomerase II inhibitor etoposide as indicated by the presence of γH2AX, a marker for unrepaired double strand breaks. Using a Comet assay, we found extensive DNA fragmentation in cells with elevated Zc3h8 expression despite high rates of proliferation and migration. Furthermore, Zc3h8 increases cellular tolerance for DNA damage and alters apoptosis. These phenotypes promote further mutation and may indicate a molecular cause for highly aggressive and progressive cancers with elevated Zc3h8 expression. These conclusions suggest a DNA damage repair and response pathway that is altered by Zc3h8 expression levels can affect multiple aspects of cellular behavior and promote oncogenic phenotypes.

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter.

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.

Influence of cellular models and individual factor in the biological response to chest CT scan exams

BackgroundWhile computed tomography (CT) exams are the major cause of medical exposure to ionising radiation, there is increasing evidence that the potential radiation-induced risks must be documented. We investigated the impact of cellular models and individual factor on the deoxyribonucleic acid double-strand breaks (DSB) recognition and repair in human fibroblasts and mammary epithelial cells exposed to current chest CT scan conditions.MethodTwelve human primary fibroblasts and four primary human mammary epithelial cell lines with different levels of radiosensitivity/susceptibility were exposed to a standard chest CT scan exam using adapted phantoms. Cells were exposed to a single helical irradiation (14.4 mGy) or to a topogram followed, after 1 min, by one single helical examination (1.1 mGy + 14.4 mGy). DSB signalling and repair was assessed through anti-γH2AX and anti-pATM immunofluorescence.ResultsChest CT scan induced a significant number of γH2AX and pATM foci. The kinetics of both biomarkers were found strongly dependent on the individual factor. The topogram may also influence the biological response of radiosensitive/susceptible fibroblasts to irradiation. Altogether, our findings show that a chest CT scan exam may result in 2 to 3 times more unrepaired DSB in cells from radiosensitive/susceptible patients.ConclusionsBoth individual and tissue factors in the recognition and repair of DSB after current CT scan exams are important. Further investigations are needed to better define the radiosensitivity/susceptibility of individual humans.

The Arabidopsis HOP2 gene has a role in preventing illegitimate connections between nonhomologous chromosome regions.

Meiotic homologous chromosomes synapse and undergo crossing over (CO). In many eukaryotes, both synapsis and crossing over require the induction of double stranded breaks (DSBs) and subsequent repair via homologous recombination. In these organisms, two key proteins are recombinases RAD51 and DMC1. Recombinase-modulators HOP2 and MND1 assist RAD51 and DMC1 and also are required for synapsis and CO. We have investigated the hop2-1 phenotype in Arabidopsis during the segregation stages of both meiosis and mitosis. Despite a general lack of synapsis during prophase I, we observed extensive, stable interconnections between nonhomologous chromosomes in diploid hop2-1 nuclei in first and second meiotic divisions. Using γH2Ax as a marker of unrepaired DSBs, we detected γH2AX foci from leptotene through early pachytene but saw no foci from mid-pachytene onward. We conclude that the bridges seen from metaphase I onward are due to mis-repaired DSBs, not unrepaired ones. Examining haploids, we found that wild type haploids produce only univalents, but hop2-1 haploids like hop2-1 diploids have illegitimate connections stable enough to produce bridged chromosomes during segregation. Our results suggest that HOP2 has a significant active role in preventing repairs that use nonhomologous chromosomes during meiosis. We also found evidence that HOP2 plays a role in preventing illegitimate repair of radiation-induced DSBs in rapidly dividing petal cells. We conclude that HOP2 in Arabidopsis plays both a positive role in promoting synapsis and a separable role in preventing DSB repair using nonhomologous chromosomes. SIGNIFICANCE STATEMENT : The fidelity of homologous recombination (HR) during meiosis is essential to the production of viable gametes and for maintaining genome integrity in vegetative cells. HOP2 is an important protein for accurate meiotic HR in plants. We have found evidence of high levels of illegitimate repairs between nonhomologous chromosomes during meiosis and in irradiated petal cells in hop2-1 mutants, suggesting a role for HOP2 beyond its established role in synapsis and crossing over.

Therapeutic Dose of Hydroxyurea-Induced Synaptic Abnormalities on the Mouse Spermatocyte.

Hydroxyurea (HU) is a widely used pharmacological therapy for sickle cell disease (SCD). However, replication stress caused by HU has been shown to inhibit premeiotic S-phase DNA, leading to reproductive toxicity in germ cells. In this study, we administered the therapeutic doses of HU (i.e., 25 and 50 mg/kg) to male mice to explore whether replication stress by HU affects pachytene spermatocytes and causes the abnormalities of homologous chromosomes pairing and recombination during prophase I of meiosis. In comparison with the control group, the proportions of spermatocyte gaps were significantly different in the experimental groups injected with 25 mg/kg (p < 0.05) and 50 mg/kg of HU (p < 0.05). Moreover, the proportions of unrepaired double-stranded breaks (DSBs) observed by γH2AX staining also corresponded to a higher HU dose with a greater number of breaks. Additionally, a reduction in the counts of recombination foci on the autosomal SCs was observed in the pachytene spermatocytes. Our results reveal that HU has some effects on synaptonemal complex (SC) formation and DSB repair which suggest possible problems in fertility. Therefore, this study provides new evidence of the mechanisms underlying HU reproductive toxicity.

The DNA damage response is required for oocyte cyst breakdown and follicle formation in mice.

Mammalian oogonia proliferate without completing cytokinesis, forming cysts. Within these, oocytes differentiate and initiate meiosis, promoting double-strand break (DSBs) formation, which are repaired by homologous recombination (HR) causing the pairing and synapsis of the homologs. Errors in these processes activate checkpoint mechanisms, leading to apoptosis. At the end of prophase I, in contrast with what is observed in spermatocytes, oocytes accumulate unrepaired DSBs. Simultaneously to the cyst breakdown, there is a massive oocyte death, which has been proposed to be necessary to enable the individualization of the oocytes to form follicles. Based upon all the above-mentioned information, we hypothesize that the apparently inefficient HR occurring in the oocytes may be a requirement to first eliminate most of the oocytes and enable cyst breakdown and follicle formation. To test this idea, we compared perinatal ovaries from control and mutant mice for the effector kinase of the DNA Damage Response (DDR), CHK2. We found that CHK2 is required to eliminate ~50% of the fetal oocyte population. Nevertheless, the number of oocytes and follicles found in Chk2-mutant ovaries three days after birth was equivalent to that of the controls. These data revealed the existence of another mechanism capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2-/- mice, implying that CHK1 regulates postnatal oocyte death. Moreover, we found that CHK1 and CHK2 functions are required for the timely breakdown of the cyst and to form follicles. Thus, we uncovered a novel CHK1 function in regulating the oocyte population in mice. Based upon these data, we propose that the CHK1- and CHK2-dependent DDR controls the number of oocytes and is required to properly break down oocyte cysts and form follicles in mammals.

Impact Of Sequencing Of Androgen Receptor-Signaling Inhibition (ARSI) And Ionizing Radiotherapy (RT) In Prostate Cancer: Importance Of Homologous Recombination (HR) Disruption

The benefit of combining ARSI with RT for the treatment of localized prostate cancer is well established. The mechanism of action has been attributed to disruption of non-homologous end-joining (NHEJ) repair via impaired DNA-dependent protein kinase (DNA-PK) function. However, clinical trials have demonstrated inconsistent benefits from combining ARSI and RT, which we hypothesized might be explained by the treatment sequence of ARSI and RT. We exposed four prostate cancer cell lines (LNCaP, LNCaP-AR, C42B, LAPC-4) to ARSI via charcoal stripped serum and/or enzalutamide, a next-generation anti-androgen. ARSI was combined with fractionated RT in three sequences: neoadjuvant, concurrent, or adjuvant. The effects of ARSI sequencing were measured with clonogenic assays, immunoblotting for signaling proteins, qPCR of AR target genes, cell cycle analysis, DNA damage and repair pathway assays, and xenograft animal studies. Clinical validation was performed by meta-analysis of individual patient data from two randomized trials comparing neoadjuvant versus adjuvant ARSI with RT. Across all prostate cancer cell lines, adjuvant sequencing of ARSI after RT was significantly more effective at killing colony forming cells (20 to 50-fold, p<0.01) and decreasing the transcription and translation of downstream AR target genes (5-fold, p<0.01). This effect was recapitulated in xenograft studies showing that adjuvant ARSI was the only regimen that slowed prostate cancer tumor growth more than RT alone. Adjuvant inhibition of the NHEJ pathway with a DNA-PK inhibitor (NU 7441) did not recapitulate the effect of adjuvant ARSI as confirmed with functional NHEJ assays. Rather, we found adjuvant ARSI treatment impaired HR by decreasing the phosphorylation of nuclear ATM, CHK2, and p53 in addition to decreasing nuclear Rad51 and increasing γH2AX staining. Adjuvant ARSI doubled (p<0.05) the proportion of cells in the G2/M phase which—in the setting of double-strand breaks (DSBs) unrepaired by HR—may explain the greater cell death seen with adjuvant treatment. Finally, we validated our preclinical findings in a meta-analysis of patient-level data, confirming that adjuvant ARSI was associated with superior progression-free survival compared to neoadjuvant/concurrent ARSI (HR: 1.25 (95%CI: 1.07-1.47), p = 0.01). We demonstrate that sequencing of ARSI and RT is of critical importance and clarify a possible mechanism. Adjuvant ARSI—rather than neoadjuvant or concurrent—is associated with the greatest tumor control with RT. The beneficial effect of adjuvant ARSI is not explained by impaired NHEJ. Rather it appears to be mediated through disruption of HR and cell cycle checkpoints in response to DNA damage, which allows prostate cancer cells to proceed to G2 and mitosis with unrepaired DSBs.

METTL3 and N6-Methyladenosine Promote Homologous Recombination-Mediated Repair of DSBs by Modulating DNA-RNA Hybrid Accumulation

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.

Oocyte Elimination Through DNA Damage Signaling from CHK1/CHK2 to p53 and p63.

Eukaryotic organisms have evolved mechanisms to prevent the accumulation of cells bearing genetic aberrations. This is especially crucial for the germline, because fecundity and fitness of progeny would be adversely affected by an excessively high mutational incidence. The process of meiosis poses unique problems for mutation avoidance because of the requirement for SPO11-induced programmed double-strand breaks (DSBs) in recombination-driven pairing and segregation of homologous chromosomes. Mouse meiocytes bearing unrepaired meiotic DSBs or unsynapsed chromosomes are eliminated before completing meiotic prophase I. In previous work, we showed that checkpoint kinase 2 (CHK2; CHEK2), a canonical DNA damage response protein, is crucial for eliminating not only oocytes defective in meiotic DSB repair (e.g., Trip13Gt mutants), but also Spo11-/- oocytes that are defective in homologous chromosome synapsis and accumulate a threshold level of spontaneous DSBs. However, rescue of such oocytes by Chk2 deficiency was incomplete, raising the possibility that a parallel checkpoint pathway(s) exists. Here, we show that mouse oocytes lacking both p53 (TRP53) and the oocyte-exclusive isoform of p63, TAp63, protects nearly all Spo11-/- and Trip13Gt/Gt oocytes from elimination. We present evidence that checkpoint kinase I (CHK1; CHEK1), which is known to signal to TRP53, also becomes activated by persistent DSBs in oocytes, and to an increased degree when CHK2 is absent. The combined data indicate that nearly all oocytes reaching a threshold level of unrepaired DSBs are eliminated by a semiredundant pathway of CHK1/CHK2 signaling to TRP53/TAp63.

An Emerging Regulatory Role for the Tumor Microenvironment in the DNA Damage Response to Double-Strand Breaks.

Radiation, alkylating agents, and platinum-based chemotherapy treatments eliminate cancer cells through the induction of excessive DNA damage. The resultant DNA damage challenges the cancer cell's DNA repair capacity. Among the different types of DNA damage induced in cells, double-strand breaks (DSB) are the most lethal if left unrepaired. Unrepaired DSBs in tumor cells exacerbate existing gene deletions, chromosome losses and rearrangements, and aberrant features that characteristically enable tumor progression, metastasis, and drug resistance. Tumor microenvironmental factors like hypoxia, inflammation, cellular metabolism, and the immune system profoundly influence DSB repair mechanisms. Here, we put into context the role of the microenvironment in governing DSB repair mechanisms.

Persistent double strand break accumulation does not precede cell death in an Olaparib-sensitive BRCA-deficient colorectal cancer cell model.

The poly (adenosine diphosphate (ADP)-ribosyl) polymerase inhibitors (PARPi) selectively kill cancer cells with BRCA1 or BRCA2 (BRCA)-mutations. It has been proposed that cell death induction after PARPi depends on unrepaired double strand breaks (DSBs) that accumulate due to the homologous recombination deficiency of BRCA-mutated cells. Such accumulation of DSBs is inferred mainly from the high levels of DNA damage markers like phosphorylated histone H2AX. Herein, we developed a model of isogenic cell lines to show that depletion of BRCA causes PARPi-triggered cell death, replication stress (phosphorylated-H2AX and 53BP1 foci), and genomic instability. However, persistent DSBs accumulation was not detected under the same experimental conditions. Hence, at least in this cellular model, the trigger for cell death in PARPi-treated BRCA-depleted samples is not the accumulation of unrepaired DSBs. Instead, cell death better correlates with a rapid and aberrant resolution of DSBs by error-prone pathways that leads to severe chromosomic aberrations. Therefore, our results suggest that in PARPi-treated BRCA-deficient cells, chromosome aberrations may dually trigger both genomic instability and cell death.