Double-strand Break Repair Research Articles

Mechanism of homology search expansion during recombinational DNA break repair in Saccharomyces cerevisiae

Homology search is a central step of DNA double-strand break (DSB) repair by homologous recombination (HR). How it operates in cells remains elusive. We developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S.cerevisiae, which revealed two main homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from local to genome-wide search by increasing the probability of assembling extensive NPFs. DSB end-tethering promotes coordinated search by opposite NPFs. Finally, an autonomous genetic element on chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals the mechanism of the progressive expansion of homology searchthat isorchestrated by chromatin organizers, long-range resection, end-tethering,andspecialized genetic elements andthatexploits the stiff NPF structure conferred by Rad51 oligomerization.

SPIN1 facilitates chemoresistance and HR repair by promoting Tip60 binding to H3K9me3

The tandem Tudor-like domain-containing protein Spindlin1 (SPIN1) is a transcriptional coactivator with critical functions in embryonic development and emerging roles in cancer. However, the involvement of SPIN1 in DNA damage repair has remained unclear. Our study shows that SPIN1 is recruited to DNA lesions through its N-terminal disordered region that binds to Poly-ADP-ribose (PAR), and facilitates homologous recombination (HR)-mediated DNA damage repair. SPIN1 promotes H3K9me3 accumulation at DNA damage sites and enhances the interaction between H3K9me3 and Tip60, thereby promoting the activation of ATM and HR repair. We also show that SPIN1 increases chemoresistance. These findings reveal a novel role for SPIN1 in the activation of H3K9me3-dependent DNA repair pathways, and suggest that SPIN1 may contribute to cancer chemoresistance by modulating the efficiency of double-strand break (DSB) repair.

Promoter Hypermethylation of the BRCA1 Gene as a Novel Biomarker for Prostate Cancer.

Prostate cancer (PCa) is recognized as one of the most common malignancies that greatly affects the male population globally. Breast cancer gene 1 (BRCA1) is an important tumor suppressor gene that plays a central role in the maintenance of genomic integrity by promoting the repair of double-strand breaks of DNA. Here, we present a pilot study to examine the promoter methylation and gene expression of the BRCA1 gene in patients with PCa in Erbil governorate, Iraq. The collection of samples took place in Erbil City, Iraq, specifically at Rizgary Hospital, PAR Hospital, and Al-Mufti's private laboratory. A total of 40 tissue samples were collected from age-matched individuals, comprising 30 pathologically confirmed PCa cases and 10 normal prostatic tissue taken from individuals who, during diagnosis, were found to be negative for PCa. Data on demographic and clinical information, such as pathological stage, age, and prostate-specific antigen (PSA) level, were gathered from the medical records. The impact of the promoter methylation was forecasted using the DNA bisulfite conversion technique and methyl-specific PCR (MSP) with specific primers for the BRCA1 promoter region. The assessment of BRCA1 expression was conducted using quantitative real-time PCR (qPCR). Among the 30 patients examined, 76.6% (23 cases) were found to have BRCA1 promoter methylation, and none of the normal tissues appeared to have DNA methylation. BRCA1 promoter methylation was positively associated with the advanced stage of disease (p=0.01) and Gleason score (p=0.007). The analysis revealed a significant downregulation of the BRCA1 gene expression in methylated tumor samples as compared to non-methylated tumors and normal tissues, suggesting the role of epigenetic silencing. To the best of our knowledge, this is the first study investigating methylation status and level of BRCA1 mRNA transcripts among PCa patients in Iraq. Our findings suggest that promoter hypermethylation of the BRCA1 gene could serve as a viable biomarker for PCa, marking a significant discovery.

Synergistic Effects of Melatonin on Radiosensitization in Carbon-ion Radiotherapy.

Despite the established antitumor effectiveness and synergistic interactions of melatonin with photon irradiation, its role in carbon-ion radiotherapy remains uncertain. This study aimed to elucidate the mechanisms and potential clinical advantages of combining exogenous melatonin therapy with carbon-ion radiotherapy. The investigation assessed the impact of combining exogenous melatonin with photon or carbon-ion irradiation on cell-cycle modulation and DNA-repair capability using the melanoma cell line B16F10. RNA sequencing and bioinformatics analysis were conducted to explore mechanisms and evaluate potential clinical benefits, with validation performed on the osteosarcoma cell line LM8. Pre-treatment with melatonin reduced the survival fraction of B16F10 and LM8 cells upon exposure to photon and carbon-ion radiation. Mechanistically, melatonin was found to inhibit G2/M arrest, preserve DNA damage, and suppress key genes involved in DNA double-strand break repair after 8 Gy carbon-ion radiation. Furthermore, RNA sequencing and bioinformatics analysis revealed favorable changes in genes associated with survival and metastasis, highlighting potential clinical significance. LM8 cells treated with melatonin exhibited increased radiosensitivity and suppression of DNA-repair proteins. The combination of exogenous melatonin not only heightened radiosensitivity and modulated hallmark tumor gene sets in vitro but also markedly suppressed the efficiency of DNA double-strand break-repair pathway, thus enhancing the cytotoxicity of carbon-ion radiotherapy.

Endogenous base damage as a driver of genomic instability in homologous recombination-deficient cancers

Homologous recombination (HR) is a high-fidelity DNA double-strand break (DSB) repair pathway. Both familial and somatic loss of function mutation(s) in various HR genes predispose to a variety of cancer types, underscoring the importance of error-free repair of DSBs in human physiology. While environmental sources of DSBs have been known, more recent studies have begun to uncover the role of endogenous base damage in leading to these breaks. Base damage repair intermediates often consist of single-strand breaks, which if left unrepaired, can lead to DSBs as the replication fork encounters these lesions. This review summarizes various sources of endogenous base damage and how these lesions are repaired. We highlight how conversion of base repair intermediates, particularly those with 5′or 3′ blocked ends, to DSBs can be a predominant source of genomic instability in HR-deficient cancers. We also discuss how endogenous base damage and ensuing DSBs can be exploited to enhance the efficacy of Poly (ADP-ribose) polymerase inhibitors (PARPi), that are widely used in the clinics for the regimen of HR-deficient cancers.

Arid1a Loss Enhances Disease Progression in a Murine Model of Osteosarcoma.

Osteosarcoma is an aggressive bone malignancy, molecularly characterized by acquired genome complexity and frequent loss of TP53 and RB1. Obtaining a molecular understanding of the initiating mutations of osteosarcomagenesis has been challenged by the difficulty of parsing between passenger and driver mutations in genes. Here, a forward genetic screen in a genetic mouse model of osteosarcomagenesis initiated by Trp53 and Rb1 conditional loss in pre-osteoblasts identified that Arid1a loss contributes to OS progression. Arid1a is a member of the canonical BAF (SWI/SNF) complex and a known tumor suppressor gene in other cancers. We hypothesized that the loss of Arid1a increases the rate of tumor progression and metastasis. Phenotypic evaluation upon in vitro and in vivo deletion of Arid1a validated this hypothesis. Gene expression and pathway analysis revealed a correlation between Arid1a loss and genomic instability, and the subsequent dysregulation of genes involved in DNA DSB or SSB repair pathways. The most significant of these transcriptional changes was a concomitant decrease in DCLRE1C. Our findings suggest that Arid1a plays a role in genomic instability in aggressive osteosarcoma and a better understanding of this correlation can help with clinical prognoses and personalized patient care.

Increased AID results in mutations at the CRLF2 locus implicated in Latin American ALL health disparities

Activation-induced cytidine deaminase (AID) is a B cell-specific mutator required for antibody diversification. However, it is also implicated in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain blood cancers is critical in assessing disease severity and treatment options. We have developed a digital PCR (dPCR) assay that allows us to quantify mutations resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this assay shows that increased AID levels in immature B cells increase genome instability at loci linked to chromosomal translocation formation. This includes the CRLF2 locus that is often involved in translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Hispanics, particularly those with Latin American ancestry. Using dPCR, we characterize the CRLF2 locus in B cell-derived genomic DNA from both Hispanic ALL patients and healthy Hispanic donors and found increased mutations in both, suggesting that vulnerability to DNA damage at CRLF2 may be driving this health disparity. Our ability to detect and quantify these mutations will potentiate future risk identification, early detection of cancers, and reduction of associated cancer health disparities.

Targeting PRMT5 enhances the radiosensitivity of tumor cells grown in vitro and in vivo

PRMT5 is a widely expressed arginine methyltransferase that regulates processes involved in tumor cell proliferation and survival. In the study described here, we investigated whether PRMT5 provides a target for tumor radiosensitization. Knockdown of PRMT5 using siRNA enhanced the radiosensitivity of a panel of cell lines corresponding to tumor types typically treated with radiotherapy. To extend these studies to an experimental therapeutic setting, the PRMT5 inhibitor LLY-283 was used. Exposure of the tumor cell lines to LLY-283 decreased PRMT5 activity and enhanced their radiosensitivity. This increase in radiosensitivity was accompanied by an inhibition of DNA double-strand break repair as determined by γH2AX foci and neutral comet analyses. For a normal fibroblast cell line, although LLY-283 reduced PRMT5 activity, it had no effect on their radiosensitivity. Transcriptome analysis of U251 cells showed that LLY-283 treatment reduced the expression of genes and altered the mRNA splicing pattern of genes involved in the DNA damage response. Subcutaneous xenografts were then used to evaluate the in vivo response to LLY-283 and radiation. Treatment of mice with LLY-283 decreased tumor PRMT5 activity and significantly enhanced the radiation-induced growth delay. These results suggest that PRMT5 is a tumor selective target for radiosensitization.

M6A-mediated lnc-OXAR promotes oxaliplatin resistance by enhancing Ku70 stability in non-alcoholic steatohepatitis-related hepatocellular carcinoma

BackgroundThe escalating prevalence of metabolic diseases has led to a rapid increase in non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (NASH-HCC). While oxaliplatin (OXA)-based hepatic arterial infusion chemotherapy (HAIC) has shown promise in advanced-stage HCC patients, its efficacy in NASH-HCC remains uncertain. This study aims to assess the effectiveness of OXA-based HAIC and elucidate the mechanisms underlying OXA resistance in NASH-HCC.MethodsThe key lncRNAs were screened through RNA-seq analysis of NASH/non-NASH and OXA-sensitive/OXA-resistant (OXA-S/R) HCC tissues. The biological functions of the lnc-OXAR (OXA resistance–related lncRNA in NASH-HCC) in NASH-HCC were verified through a series of in vitro and in vivo experiments. The molecular mechanism of lnc-OXAR was elucidated by fluorescence in situ hybridization, immunoprecipitation-mass spectrometry (FISH), Immunoprecipitation-Mass Spectrometry (IP-MS), RNA pulldown, RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and a dual-luciferase reporter assay.ResultsNASH-HCC exhibited reduced responsiveness to OXA-based HAIC compared to non-NASH HCC. We identified and validated a novel transcript namedlnc-OXAR, which played a crucial role in conferring OXA resistance to NASH-HCC. Inhibition of lnc-OXAR suppressed HCC cell growth and restored OXA sensitivity both in NASH-HCC mouse models and in vitro. Mechanistically, lnc-OXAR recruited Ku70 and cystatin A (CSTA), preventing Ku70 degradation and facilitating DNA double-strand break (DSB) repair, thereby promoting OXA resistance in NASH-HCC. Additionally, WTAP-mediated m6A modification enhanced the stability of lnc-OXAR in an IGF2BP2-dependent manner. Notably, silencing lnc-OXAR significantly enhanced the response to OXA in patient-derived xenograft (PDX) models derived from NASH-HCC.ConclusionsThe reduced responsiveness of NASH-HCC to OXA treatment can be attributed to the upregulation of lnc-OXAR. Our findings provide a rationale for stratifying HCC patients undergoing OXA-based HAIC based on etiology. Lnc-OXAR holds promise as a novel target for overcoming OXA resistance in NASH-HCC and improving prognosis.

M6A demethylase OsALKBH5 is required for double-strand break formation and repair by affecting mRNA stability in rice meiosis.

N6-methyladenosine (m6A) RNA modification is the most prevalent messenger RNA (mRNA) modification in eukaryotes and plays critical roles in the regulation of gene expression. m6A is a reversible RNA modification that is deposited by methyltransferases (writers) and removed by demethylases (erasers). The function of m6A erasers in plants is highly diversified and their roles in cereal crops, especially in reproductive development essential for crop yield, are largely unknown. Here, we demonstrate that rice OsALKBH5 acts as an m6A demethylase required for the normal progression of male meiosis. OsALKBH5 is a nucleo-cytoplasmic protein, highly enriched in rice anthers during meiosis, that associates with P-bodies and exon junction complexes, suggesting that it is involved in regulating mRNA processing and abundance. Mutations of OsALKBH5 cause reduced double-strand break (DSB) formation, severe defects in DSB repair, and delayed meiotic progression, leading to complete male sterility. Transcriptome analysis and m6A profiling indicate that OsALKBH5-mediated m6A demethylation stabilizes the mRNA level of multiple meiotic genes directly or indirectly, including several genes that regulate DSB formation and repair. Our study reveals the indispensable role of m6A metabolism in post-transcriptional regulation of meiotic progression in rice.

Genome Editing of Mammalian Cells Through RNA Transcript-Mediated Homologous Recombination Repair.

Double-stranded break (DSB) repair of eukaryotic DNA is mainly accomplished by nonhomologous end joining and homologous recombination (HR). Providing exogenous templates during HR repair can result in the editing of target genes, which is the central mechanism of the well-established clustered regularly interspaced short palindromic repeats (CRISPR) gene editing system. Currently, exogenous templates are mainly DNA molecules, which can provoke a cellular immune response within the cell. In order to verify the feasibility of RNA molecules as repair templates for HR in mammalian cell genome editing, we fused RNA template molecules to the 3'-end of single guide RNA (sgRNA), so that the sgRNA and the homologous template RNA form a single RNA molecule. The results show this construct can be used as a repair template to achieve target gene editing in mammalian cells. In addition, the factors influencing HR mediated by RNA template molecules were investigated, and it was found that increasing the length of homologous arms and inducing an R-loop near the DSBcan effectively promote HR repair. Furthermore, intracellular homologous chromosomes may compete with exogenous RNA templates. The findings in this article provide a reference for the utilization of RNA template molecules to mediate target gene editing in eukaryotic cells, as well as a basis for the study of the mechanism by which RNA molecules mediate the repair of DSBs.

SMARCA2 and SMARCA4 Participate in DNA Damage Repair.

The switching/sucrose non-fermentable (SWI/SNF) Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A (SMARCA) member 2 and member 4 (SMARCA2/4) are paralogs and act as the key enzymatic subunits in the SWI/SNF complex for chromatin remodeling. However, the role of SMARCA2/4 in DNA damage response remains unclear. Laser microirradiation assays were performed to examine the key domains of SMARCA2/4 for the relocation of the SWI/SNF complex to DNA lesions. To examine the key factors that mediate the recruitment of SMARCA2/4, the relocation of SMARCA2/4 to DNA lesions was examined in HeLa cells treated with inhibitors of Ataxia-telangiectasia-mutated (ATM), Ataxia telangiectasia and Rad3-related protein (ATR), CREB-binding protein (CBP) and its homologue p300 (p300/CBP), or Poly (ADP-ribose) polymerase (PARP) 1/2 as well as in H2AX-deficient HeLa cells. Moreover, by concomitantly suppressing SMARCA2/4 with the small molecule inhibitor FHD286 or Compound 14, the function of SMARCA2/4 in Radiation sensitive 51 (RAD51) foci formation and homologous recombination repair was examined. Finally, using a colony formation assay, the synergistic effect of PARP inhibitors and SMARCA2/4 inhibitors on the suppression of tumor cell growth was examined. We show that SMARCA2/4 relocate to DNA lesions in response to DNA damage, which requires their ATPase activities. Moreover, these ATPase activities are also required for the relocation of other subunits in the SWI/SNF complex to DNA lesions. Interestingly, the relocation of SMARCA2/4 is independent of γH2AX, ATM, ATR, p300/CBP, or PARP1/2, indicating that it may directly recognize DNA lesions as a DNA damage sensor. Lacking SMARCA2/4 prolongs the retention of γH2AX, Ring Finger Protein 8 (RNF8) and Breast cancer susceptibility gene 1 (BRCA1) at DNA lesions and impairs RAD51-dependent homologous recombination repair. Furthermore, the treatment of an SMARCA2/4 inhibitor sensitizes tumor cells to PARP inhibitor treatment. This study reveals SMARCA2/4 as a DNA damage repair factor for double-strand break repair.

Tau beyond Tangles: DNA Damage Response and Cytoskeletal Protein Crosstalk on Neurodegeneration.

Neurons in the brain are continuously exposed to various sources of DNA damage. Although the mechanisms of DNA damage repair in mitotic cells have been extensively characterized, the repair pathways in post-mitotic neurons are still largely elusive. Moreover, inaccurate repair can result in deleterious mutations, including deletions, insertions, and chromosomal translocations, ultimately compromising genomic stability. Since neurons are terminally differentiated cells, they cannot employ homologous recombination (HR) for double-strand break (DSB) repair, suggesting the existence of neuron-specific repair mechanisms. Our research has centered on the microtubule-associated protein tau (MAPT), a crucial pathological protein implicated in neurodegenerative diseases, and its interplay with neurons' DNA damage response (DDR). This review aims to provide an updated synthesis of the current understanding of the complex interplay between DDR and cytoskeletal proteins in neurons, with a particular focus on the role of tau in neurodegenerative disorders.

DNA cytosine methyltransferases differentially regulate genome-wide hypermutation and interhomolog recombination in Trichoderma reesei meiosis.

Trichoderma reesei is an economically important enzyme producer with several unique meiotic features. spo11, the initiator of meiotic double-strand breaks (DSBs) in most sexual eukaryotes, is dispensable for T. reesei meiosis. T. reesei lacks the meiosis-specific recombinase Dmc1. Rad51 and Sae2, the activator of the Mre11 endonuclease complex, promote DSB repair and chromosome synapsis in wild-type and spo11Δ meiosis. DNA methyltransferases (DNMTs) perform multiple tasks in meiosis. Three DNMT genes (rid1, dim2and dimX) differentially regulate genome-wide cytosine methylation and C:G-to-T:A hypermutations in different chromosomal regions. We have identified two types of DSBs: type I DSBs require spo11 or rid1 for initiation, whereas type II DSBs do not rely on spo11 and rid1 for initiation. rid1 (but not dim2) is essential for Rad51-mediated DSB repair and normal meiosis. rid1 and rad51 exhibit a locus heterogeneity (LH) relationship, in which LH-associated proteins often regulate interconnectivity in protein interaction networks. This LH relationship can be suppressed by deleting dim2 in a haploid rid1Δ (but not rad51Δ) parental strain, indicating that dim2 and rid1 share a redundant function that acts earlier than rad51 during early meiosis. In conclusion, our studies provide the first evidence of the involvement of DNMTs during meiotic initiation and recombination.

Human translesion DNA polymerases ι and κ mediate tolerance to temozolomide in MGMT-deficient glioblastoma cells.

Glioblastoma (GBM) is a highly aggressive brain tumor associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing temozolomide (TMZ). Resistance to TMZ is, however, a major challenge. Previous work from our group has identified candidate genes linked to TMZ resistance, including genes encoding translesion synthesis (TLS) DNA polymerases iota (Polɩ) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of Polɩ and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding Polɩ and Polκ, respectively, and assess their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant decrease in viability following TMZ treatment compared to parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed increased cell cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction of γH2AX foci and flow cytometry data. This implies that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (double-strand break repair pathway inhibitor) further reduced the cell viability and increased DNA damage and γH2AX positive cells in TLS KO cells, but not in parental cells. These findings underscore the crucial roles of Polɩ and Polκ in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. Targeting these TLS enzymes, along with double-strand break DNA repair inhibition, could, therefore, provide a promising strategy to enhance TMZ's effectiveness in treating GBM.

Multifunctional Glycopeptide-Based Hydrogel via Dual-Modulation for the Prevention and Repair of Radiation-Induced Skin Injury.

The radiation-induced skin injury (RISI) remains a great challenge for clinical wound management and care after radiotherapy, as patients will suffer from the acute radiation injury and long-term chronic inflammatory damage during the treatment. The excessive ROS in the early acute stage and prolonged inflammatory response in the late healing process always hinder therapeutic efficiency. Herein, we developed an extracellular matrix (ECM)-mimetic multifunctional glycopeptide hydrogel (oCP@As) to promote and accelerate RISI repair via a dual-modulation strategy in different healing stages. The oCP@As hydrogel not only can form an ECM-like nanofiber structure through the Schiff base reaction but also exhibits ROS scavenging and DNA double-strand break repair abilities, which can effectively reduce the acute radiation damage. Meanwhile, the introduction of oxidized chondroitin sulfate, which is the ECM polysaccharide-like component, enables regulation of the inflammatory response by adsorption of inflammatory factors, accelerating the repair of chronic inflammatory injury. The animal experiments demonstrated that oCP@As can significantly weaken RISI symptoms, promote epidermal tissue regeneration and angiogenesis, and reduce pro-inflammatory cytokine expression. Therefore, this multifunctional glycopeptide hydrogel dressing can effectively attenuate RISI symptoms and promote RISI healing, showing great potential for clinical applications in radiotherapy protection and repair.

A Genomewide Evolution-Based CRISPR/Cas9 with Donor-Free (GEbCD) for Developing Robust and Productive Industrial Yeast.

Developing more robust and productive industrial yeast is crucial for high-efficiency biomanufacturing. However, the challenges posed by the long time required and the low abundance of mutations generated through genomewide evolutionary engineering hinder the development and optimization of desired hosts for industrial applications. To address these issues, we present a novel solution called the Genomewide Evolution-based CRISPR/Cas with Donor-free (GEbCD) system, in which nonhomologous-end-joining (NHEJ) repair can accelerate the acquisition of highly abundant yeast mutants. Together with modified rad52 of the DNA double-strand break repair in Saccharomyces cerevisiae, a hypermutation host was obtained with a 400-fold enhanced mutation ability. Under multiple environmental stresses the system could rapidly generate millions of mutants in a few rounds of iterative evolution. Using high-throughput screening, an industrial S. cerevisiae SISc-Δrad52-G4-72 (G4-72) was obtained that is strongly robust and has higher productivity. G4-72 grew stably and produced ethanol efficiently in multiple-stress environments, e.g. high temperature and high osmosis. In a pilot-scale fermentation with G4-72, the fermentation temperature was elevated by 8 °C and ethanol production was increased by 6.9% under the multiple stresses posed by the industrial fermentation substrate. Overall, the GEbCD system presents a powerful tool to rapidly generate abundant mutants and desired hosts, and offers a novel strategy for optimizing microbial chassis with regard to demands posed in industrial applications.

Meiotic double-strand break repair DNA synthesis tracts in Arabidopsis thaliana.

We report here the successful labelling of meiotic prophase I DNA synthesis in the flowering plant, Arabidopsis thaliana. Incorporation of the thymidine analogue, EdU, enables visualisation of the footprints of recombinational repair of programmed meiotic DNA double-strand breaks (DSB), with ~400 discrete, SPO11-dependent, EdU-labelled chromosomal foci clearly visible at pachytene and later stages of meiosis. This number equates well with previous estimations of 200-300 DNA double-strand breaks per meiosis in Arabidopsis, confirming the power of this approach to detect the repair of most or all SPO11-dependent meiotic DSB repair events. The chromosomal distribution of these DNA-synthesis foci accords with that of early recombination markers and MLH1, which marks Class I crossover sites. Approximately 10 inter-homologue cross-overs (CO) have been shown to occur in each Arabidopsis male meiosis and, athough very probably under-estimated, an equivalent number of inter-homologue gene conversions (GC) have been described. Thus, at least 90% of meiotic recombination events, and very probably more, have not previously been accessible for analysis. Visual examination of the patterns of the foci on the synapsed pachytene chromosomes corresponds well with expectations from the different mechanisms of meiotic recombination and notably, no evidence for long Break-Induced Replication DNA synthesis tracts was found. Labelling of meiotic prophase I, SPO11-dependent DNA synthesis holds great promise for further understanding of the molecular mechanisms of meiotic recombination, at the heart of reproduction and evolution of eukaryotes.

Identification of 6-Anilino Imidazo[4,5-c]pyridin-2-ones as Selective DNA-Dependent Protein Kinase Inhibitors and Their Application as Radiosensitizers.

The dominant role of non-homologous end-joining in the repair of radiation-induced double-strand breaks identifies DNA-dependent protein kinase (DNA-PK) as an excellent target for the development of radiosensitizers. We report the discovery of a new class of imidazo[4,5-c]pyridine-2-one DNA-PK inhibitors. Structure-activity studies culminated in the identification of 78 as a nM DNA-PK inhibitor with excellent selectivity for DNA-PK compared to related phosphoinositide 3-kinase (PI3K) and PI3K-like kinase (PIKK) families and the broader kinome, and displayed DNA-PK-dependent radiosensitization of HAP1 cells. Compound 78 demonstrated robust radiosensitization of a broad range of cancer cells in vitro, displayed high oral bioavailability, and sensitized colorectal carcinoma (HCT116/54C) and head and neck squamous cell carcinoma (UT-SCC-74B) tumor xenografts to radiation. Compound 78 also provided substantial tumor growth inhibition of HCT116/54C tumor xenografts in combination with radiation. Compound 78 represents a new, potent, and selective class of DNA-PK inhibitors with significant potential as radiosensitizers for cancer treatment.

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.