Targeted nuclear irradiation with a proton microbeam induces oxidative DNA base damage and triggers the recruitment of DNA glycosylases OGG1 and NTH1.

DNA is the major target of radiation therapy of malignant tumors. Ionizing radiation (IR) induces a variety of DNA lesions, including chemically modified bases and strand breaks. The use of proton beam therapy for cancer treatment is ramping up, as it is expected to reduce normal tissue damage. Thus, it is important to understand the molecular mechanisms of recognition, signaling, and repair of DNA damage induced by protons in the perspective of assessing not only the risk associated with human exposure to IR but also the possibility to improve the efficacy of therapy. Here, we used targeted irradiation of nuclear regions of living cells with controlled number of protons at a high spatio-temporal resolution to detect the induced base lesions and characterize the recruitment kinetics of the specific DNA glycosylases to DNA damage sites. We show that localized irradiation with 4 MeV protons induces, in addition to DNA double strand breaks (DSBs), the oxidized bases 7,8-dihydro-8-oxoguanine (8-oxoG) and thymine glycol (TG) at the site of irradiation. Consistently, the DNA glycosylases OGG1 and NTH1, capable of excising 8-oxoG and TG, respectively, and initiating the base excision repair (BER) pathway, are recruited to the site of damage. To our knowledge, this is the first direct evidence indicating that proton microbeams induce oxidative base damage, and thus implicating BER in the repair of DNA lesions induced by protons.

Spatio-temporal dynamics of the DNA glycosylase OGG1 in finding and processing 8-oxoguanine.

OGG1 is the DNA glycosylase responsible for the removal of the oxidative lesion 8-oxoguanine (8-oxoG) from DNA. The recognition of this lesion by OGG1 is a complex process that involves scanning the DNA for the presence of 8-oxoG, followed by recognition and lesion removal. Structural data have shown that OGG1 evolves through different stages of conformation onto the DNA, corresponding to elementary steps of the 8-oxoG recognition and extrusion from the double helix. Single-molecule studies of OGG1 on naked DNA have shown that OGG1 slides in persistent contact with the DNA, displaying different binding states probably corresponding to the different conformation stages. However, in cells, the DNA is not naked and OGG1 has to navigate into a complex and highly crowded environment within the nucleus. To ensure rapid detection of 8-oxoG, OGG1 alternates between 3D diffusion and sliding along the DNA. This process is regulated by the local chromatin state but also by protein co-factors that could facilitate the detection of oxidized lesions. We will review here the different methods that have been used over the last years to better understand how OGG1 detects and process 8-oxoG lesions.

Requirement of transcription-coupled nucleotide excision repair for the removal of a specific type of oxidatively induced DNA damage.

Accumulation of DNA damage resulting from reactive oxygen species was proposed to cause neurological and degenerative disease in patients, deficient in nucleotide excision repair (NER) or its transcription-coupled subpathway (TC-NER). Here, we assessed the requirement of TC-NER for the repair of specific types of oxidatively generated DNA modifications. We incorporated synthetic 5',8-cyclo-2'-deoxypurine nucleotides (cyclo-dA, cyclo-dG) and thymine glycol (Tg) into an EGFP reporter gene to measure transcription-blocking potentials of these modifications in human cells. Using null mutants, we further identified the relevant DNA repair components by a host cell reactivation approach. The results indicated that NTHL1-initiated base excision repair is by far the most efficient pathway for Tg. Moreover, Tg was efficiently bypassed during transcription, which effectively rules out TC-NER as an alternative repair mechanism. In a sharp contrast, both cyclopurine lesions robustly blocked transcription and were repaired by NER, wherein the specific TC-NER components CSB/ERCC6 and CSA/ERCC8 were as essential as XPA. Instead, repair of classical NER substrates, cyclobutane pyrimidine dimer and N-(deoxyguanosin-8-yl)-2-acetylaminofluorene, occurred even when TC-NER was disrupted. The strict requirement of TC-NER highlights cyclo-dA and cyclo-dG as candidate damage types, accountable for cytotoxic and degenerative responses in individuals affected by genetic defects in this pathway.

Identification of key residues of the DNA glycosylase OGG1 controlling efficient DNA sampling and recruitment to oxidized bases in living cells.

The DNA-glycosylase OGG1 oversees the detection and clearance of the 7,8-dihydro-8-oxoguanine (8-oxoG), which is the most frequent form of oxidized base in the genome. This lesion is deeply buried within the double-helix and its detection requires careful inspection of the bases by OGG1 via a mechanism that remains only partially understood. By analyzing OGG1 dynamics in the nucleus of living human cells, we demonstrate that the glycosylase constantly samples the DNA by rapidly alternating between diffusion within the nucleoplasm and short transits on the DNA. This sampling process, that we find to be tightly regulated by the conserved residue G245, is crucial for the rapid recruitment of OGG1 at oxidative lesions induced by laser micro-irradiation. Furthermore, we show that residues Y203, N149 and N150, while being all involved in early stages of 8-oxoG probing by OGG1 based on previous structural data, differentially regulate the sampling of the DNA and recruitment to oxidative lesions.

OGG1 competitive inhibitors show important off-target effects by directly inhibiting efflux pumps and disturbing mitotic progression.

One of the most abundant DNA lesions induced by Reactive oxygen species (ROS) is 8-oxoG, a highly mutagenic lesion that compromises genetic instability when not efficiently repaired. 8-oxoG is specifically recognized by the DNA-glycosylase OGG1 that excises the base and initiates the Base Excision Repair pathway (BER). Furthermore, OGG1 has not only a major role in DNA repair but it is also involved in transcriptional regulation. Cancer cells are particularly exposed to ROS, thus challenging their capacity to process oxidative DNA damage has been proposed as a promising therapeutic strategy for cancer treatment. Two competitive inhibitors of OGG1 (OGG1i) have been identified, TH5487 and SU0268, which bind to the OGG1 catalytic pocket preventing its fixation to the DNA. Early studies with these inhibitors show an enhanced cellular sensitivity to cytotoxic drugs and a reduction in the inflammatory response. Our study uncovers two unreported off-targets effects of these OGG1i that are independent of OGG1. In vitro and in cellulo approaches have unveiled that OGG1i TH5487 and SU0268, despite an unrelated molecular structure, are able to inhibit some members of the ABC family transporters, in particular ABC B1 (MDR1) and ABC G2 (BCRP). The inhibition of these efflux pumps by OGG1 inhibitors results in a higher intra-cellular accumulation of various fluorescent probes and drugs, and largely contributes to the enhanced cytotoxicity observed when the inhibitors are combined with cytotoxic agents. Furthermore, we found that SU0268 has an OGG1-independent anti-mitotic activity-by interfering with metaphase completion-resulting in a high cellular toxicity. These two off-target activities are observed at concentrations of OGG1i that are normally used for in vivo studies. It is thus critical to consider these previously unreported non-specific effects when interpreting studies using TH5487 and SU0268 in the context of OGG1 inhibition. Additionally, our work highlights the persistent need for new specific inhibitors of the enzymatic activity of OGG1.

Foetal exposure to the bisphenols BADGE and BPAF impairs meiosis through DNA oxidation in mouse ovaries.

Many endocrine disruptors have been proven to impair the meiotic process which is required for the production of healthy gametes. Bisphenol A is emblematic of such disruptors, as it impairs meiotic prophase I and causes oocyte aneuploidy following in utero exposure. However, the mechanisms underlying these deleterious effects remain poorly understood. Furthermore, the increasing use of BPA alternatives raises concerns for public health. Here, we investigated the effects of foetal exposure to two BPA alternatives, bisphenol A Diglycidyl Ether (BADGE) and bisphenol AF (BPAF), on oogenesis in mice. These compounds delay meiosis initiation, increase the number of MLH1 foci per cell and induce oocyte aneuploidy. We further demonstrate that these defects are accompanied by changes in gene expression in foetal premeiotic germ cells and aberrant mRNA splicing of meiotic genes. We observed an increase in DNA oxidation after exposure to BPA alternatives. Specific induction of oxidative DNA damage during foetal germ cell differentiation causes similar defects during oogenesis, as observed in 8-oxoguanine DNA Glycosylase (OGG1)-deficient mice or after in utero exposure to potassium bromate (KBrO3), an inducer of oxidative DNA damage. The supplementation of BPA alternatives with N-acetylcysteine (NAC) counteracts the effects of bisphenols on meiosis. Together, our results propose oxidative DNA lesion as an event that negatively impacts female meiosis with major consequences on oocyte quality. This could be a common mechanism of action for numerous environmental pro-oxidant pollutants, and its discovery, could lead to reconsider the adverse effect of bisphenol mixtures that are simultaneously present in our environment.

Loss of CD24 promotes radiation­ and chemo­resistance by inducing stemness properties associated with a hybrid E/M state in breast cancer cells.

Cancer stem cells (CSCs) serve an essential role in failure of conventional antitumor therapy. In breast cancer, CD24­/low/CD44+ phenotype and high aldehyde dehydrogenase activity are associated with CSC subtypes. Furthermore, CD24­/low/CD44+ pattern is also characteristic of mesenchymal cells generated by epithelial­mesenchymal transition (EMT). CD24 is a surface marker expressed in numerous types of tumor, however, its biological functions and role in cancer progression and treatment resistance remain poorly documented. Loss of CD24 expression in breast cancer cells is associated with radiation resistance and control of oxidative stress. Reactive oxygen species (ROS) mediate the effects of anticancer drugs as well as ionizing radiation; therefore, the present study investigated if CD24 mediates radiation­ and chemo­resistance of breast cancer cells. Using a HMLE breast cancer cell model, CD24 expression has been artificially modulated and it was observed that loss of CD24 expression induced stemness properties associated with acquisition of a hybrid E/M phenotype. CD24­/low cells were more radiation­ and chemo­resistant than CD24+ cells. The resistance was associated with lower levels of ROS; CD24 controlled ROS levels via regulation of mitochondrial function independently of antioxidant activity. Together, these results suggested a key role of CD24 in de­differentiation of breast cancer cells and promoting acquisition of therapeutic resistance properties.

Lamin B1 sequesters 53BP1 to control its recruitment to DNA damage.

Double-strand breaks (DSBs) are harmful lesions and a major cause of genome instability. Studies have suggested a link between the nuclear envelope and the DNA damage response. Here, we show that lamin B1, a major component of the nuclear envelope, interacts directly with 53BP1 protein, which plays a pivotal role in the DSB repair. This interaction is dissociated after DNA damage. Lamin B1 overexpression impedes 53BP1 recruitment to DNA damage sites and leads to a persistence of DNA damage, a defect in nonhomologous end joining and an increased sensitivity to DSBs. The identification of interactions domains between lamin B1 and 53BP1 allows us to demonstrate that the defect of 53BP1 recruitment and the DSB persistence upon lamin B1 overexpression are due to sequestration of 53BP1 by lamin B1. This study highlights lamin B1 as a factor controlling the recruitment of 53BP1 to DNA damage sites upon injury.

Lost in the Crowd: How Does Human 8-Oxoguanine DNA Glycosylase 1 (OGG1) Find 8-Oxoguanine in the Genome?

The most frequent DNA lesion resulting from an oxidative stress is 7,8-dihydro-8-oxoguanine (8-oxoG). 8-oxoG is a premutagenic base modification due to its capacity to pair with adenine. Thus, the repair of 8-oxoG is critical for the preservation of the genetic information. Nowadays, 8-oxoG is also considered as an oxidative stress-sensor with a putative role in transcription regulation. In mammalian cells, the modified base is excised by the 8-oxoguanine DNA glycosylase (OGG1), initiating the base excision repair (BER) pathway. OGG1 confronts the massive challenge that is finding rare occurrences of 8-oxoG among a million-fold excess of normal guanines. Here, we review the current knowledge on the search and discrimination mechanisms employed by OGG1 to find its substrate in the genome. While there is considerable data from in vitro experiments, much less is known on how OGG1 is recruited to chromatin and scans the genome within the cellular nucleus. Based on what is known of the strategies used by proteins searching for rare genomic targets, we discuss the possible scenarios allowing the efficient detection of 8-oxoG by OGG1.

MasterPATH: network analysis of functional genomics screening data.

BACKGROUND: Functional genomics employs several experimental approaches to investigate gene functions. High-throughput techniques, such as loss-of-function screening and transcriptome profiling, allow to identify lists of genes potentially involved in biological processes of interest (so called hit list). Several computational methods exist to analyze and interpret such lists, the most widespread of which aim either at investigating of significantly enriched biological processes, or at extracting significantly represented subnetworks. RESULTS: Here we propose a novel network analysis method and corresponding computational software that employs the shortest path approach and centrality measure to discover members of molecular pathways leading to the studied phenotype, based on functional genomics screening data. The method works on integrated interactomes that consist of both directed and undirected networks - HIPPIE, SIGNOR, SignaLink, TFactS, KEGG, TransmiR, miRTarBase. The method finds nodes and short simple paths with significant high centrality in subnetworks induced by the hit genes and by so-called final implementers - the genes that are involved in molecular events responsible for final phenotypic realization of the biological processes of interest. We present the application of the method to the data from miRNA loss-of-function screen and transcriptome profiling of terminal human muscle differentiation process and to the gene loss-of-function screen exploring the genes that regulates human oxidative DNA damage recognition. The analysis highlighted the possible role of several known myogenesis regulatory miRNAs (miR-1, miR-125b, miR-216a) and their targets (AR, NR3C1, ARRB1, ITSN1, VAV3, TDGF1), as well as linked two major regulatory molecules of skeletal myogenesis, MYOD and SMAD3, to their previously known muscle-related targets (TGFB1, CDC42, CTCF) and also to a number of proteins such as C-KIT that have not been previously studied in the context of muscle differentiation. The analysis also showed the role of the interaction between H3 and SETDB1 proteins for oxidative DNA damage recognition. CONCLUSION: The current work provides a systematic methodology to discover members of molecular pathways in integrated networks using functional genomics screening data. It also offers a valuable instrument to explain the appearance of a set of genes, previously not associated with the process of interest, in the hit list of each particular functional genomics screening.

Chromatin recruitment of OGG1 requires cohesin and mediator and is essential for efficient 8-oxoG removal.

One of the most abundant DNA lesions induced by oxidative stress is the highly mutagenic 8-oxoguanine (8-oxoG), which is specifically recognized by 8-oxoguanine DNA glycosylase 1 (OGG1) to initiate its repair. How DNA glycosylases find small non-helix-distorting DNA lesions amongst millions of bases packaged in the chromatin-based architecture of the genome remains an open question. Here, we used a high-throughput siRNA screening to identify factors involved in the recognition of 8-oxoG by OGG1. We show that cohesin and mediator subunits are required for re-localization of OGG1 and other base excision repair factors to chromatin upon oxidative stress. The association of OGG1 with euchromatin is necessary for the removal of 8-oxoG. Mediator subunits CDK8 and MED12 bind to chromatin and interact with OGG1 in response to oxidative stress, suggesting they participate in the recruitment of the DNA glycosylase. The oxidative stress-induced association between the cohesin and mediator complexes and OGG1 reveals an unsuspected function of those complexes in the maintenance of genomic stability.

Lensless microscopy platform for single cell and tissue visualization.

Today, 3D imaging techniques are emerging, not only as a new tool in early drug discovery but also for the development of potential therapeutics to treat disease. Particular efforts are directed towards in vivo physiology to avoid perturbing the system under study. Here, we assess non-invasive 3D lensless imaging and its impact on cell behavior and analysis. We test our concept on various bio-applications and present here the first results. The microscopy platform based on in-holography provides large fields of view images (several mm2 compared to several hundred µm2) with sub-micrometer spatial resolution. 3D image reconstructions are achieved using back propagation functions post-processing.

Human hematopoietic stem/progenitor cells display reactive oxygen species-dependent long-term hematopoietic defects after exposure to low doses of ionizing radiations.

Hematopoietic stem cells are responsible for life-long blood cell production and are highly sensitive to exogenous stresses. The effects of low doses of ionizing radiations on radiosensitive tissues such as the hematopoietic tissue are still unknown despite their increasing use in medical imaging. Here, we study the consequences of low doses of ionizing radiations on differentiation and self-renewal capacities of human primary hematopoietic stem/progenitor cells (HSPC). We found that a single 20 mGy dose impairs the hematopoietic reconstitution potential of human HSPC but not their differentiation properties. In contrast to high irradiation doses, low doses of irradiation do not induce DNA double strand breaks in HSPC but, similar to high doses, induce a rapid and transient increase of reactive oxygen species (ROS) that promotes activation of the p38MAPK pathway. HSPC treatment with ROS scavengers or p38MAPK inhibitor prior exposure to 20 mGy irradiation abolishes the 20 mGy-induced defects indicating that ROS and p38MAPK pathways are transducers of low doses of radiation effects. Taken together, these results show that a 20 mGy dose of ionizing radiation reduces the reconstitution potential of HSPC suggesting an effect on the self-renewal potential of human hematopoietic stem cells and pinpointing ROS or the p38MAPK as therapeutic targets. Inhibition of ROS or the p38MAPK pathway protects human primary HSPC from low-dose irradiation toxicity.

Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage.

Repair of two major forms of DNA damage, single strand breaks and base modifications, are dependent on XRCC1. XRCC1 orchestrates these repair processes by temporally and spatially coordinating interactions between several other repair proteins. Here we show that XRCC1 contains a central DNA binding domain (CDB, residues 219-415) encompassing its first BRCT domain. In contrast to the N-terminal domain of XRCC1, which has been reported to mediate damage sensing in vitro, we demonstrate that the DNA binding module identified here lacks binding specificity towards DNA containing nicks or gaps. Alanine substitution of residues within the CDB of XRCC1 disrupt DNA binding in vitro and lead to a significant reduction in XRCC1 retention at DNA damage sites without affecting initial recruitment. Interestingly, reduced retention at sites of DNA damage is associated with an increased rate of repair. These findings suggest that DNA binding activity of XRCC1 plays a significant role in retention at sites of damage and the rate at which damage is repaired.

The transcription-coupled DNA repair-initiating protein CSB promotes XRCC1 recruitment to oxidative DNA damage.

Transcription-coupled nucleotide excision repair factor Cockayne syndrome protein B (CSB) was suggested to function in the repair of oxidative DNA damage. However thus far, no clear role for CSB in base excision repair (BER), the dedicated pathway to remove abundant oxidative DNA damage, could be established. Using live cell imaging with a laser-assisted procedure to locally induce 8-oxo-7,8-dihydroguanine (8-oxoG) lesions, we previously showed that CSB is recruited to these lesions in a transcription-dependent but NER-independent fashion. Here we showed that recruitment of the preferred 8-oxoG-glycosylase 1 (OGG1) is independent of CSB or active transcription. In contrast, recruitment of the BER-scaffolding protein, X-ray repair cross-complementing protein 1 (XRCC1), to 8-oxoG lesions is stimulated by CSB and transcription. Remarkably, recruitment of XRCC1 to BER-unrelated single strand breaks (SSBs) does not require CSB or transcription. Together, our results suggest a specific transcription-dependent role for CSB in recruiting XRCC1 to BER-generated SSBs, whereas XRCC1 recruitment to SSBs generated independently of BER relies predominantly on PARP activation. Based on our results, we propose a model in which CSB plays a role in facilitating BER progression at transcribed genes, probably to allow XRCC1 recruitment to BER-intermediates masked by RNA polymerase II complexes stalled at these intermediates.

Mitochondrial maintenance under oxidative stress depends on mitochondrially localised α-OGG1.

Accumulation of 8-oxoguanine (8-oxoG) in mitochondrial DNA and mitochondrial dysfunction have been observed in cells deficient for the DNA glycosylase OGG1 when exposed to oxidative stress. In human cells, up to eight mRNAs for OGG1 can be generated by alternative splicing and it is still unclear which of them codes for the protein that ensures the repair of 8-oxoG in mitochondria. Here, we show that the α-OGG1 isoform, considered up to now to be exclusively nuclear, has a functional mitochondrial-targeting sequence and is imported into mitochondria. We analyse the sub-mitochondrial localisation of α-OGG1 with unprecedented resolution and show that this DNA glycosylase is associated with DNA in mitochondrial nucleoids. We show that the presence of α-OGG1 inside mitochondria and its enzymatic activity are required to preserve the mitochondrial network in cells exposed to oxidative stress. Altogether, these results unveil a new role of α-OGG1 in the mitochondria and indicate that the same isoform ensures the repair of 8-oxoG in both nuclear and mitochondrial genomes. The activity of α-OGG1 in mitochondria is sufficient for the recovery of organelle function after oxidative stress.

Incorporating interaction networks into the determination of functionally related hit genes in genomic experiments with Markov random fields.

MOTIVATION: Incorporating gene interaction data into the identification of 'hit' genes in genomic experiments is a well-established approach leveraging the 'guilt by association' assumption to obtain a network based hit list of functionally related genes. We aim to develop a method to allow for multivariate gene scores and multiple hit labels in order to extend the analysis of genomic screening data within such an approach. RESULTS: We propose a Markov random field-based method to achieve our aim and show that the particular advantages of our method compared with those currently used lead to new insights in previously analysed data as well as for our own motivating data. Our method additionally achieves the best performance in an independent simulation experiment. The real data applications we consider comprise of a survival analysis and differential expression experiment and a cell-based RNA interference functional screen. AVAILABILITY AND IMPLEMENTATION: We provide all of the data and code related to the results in the paper. CONTACT: sean.j.robinson@utu.fi or laurent.guyon@cea.fr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

XPC deficiency is related to APE1 and OGG1 expression and function.

Oxidative DNA damage is considered to be a major cause of neurodegeneration and internal tumors observed in syndromes that result from nucleotide excision repair (NER) deficiencies, such as Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS). Recent evidence has shown that NER aids in removing oxidized DNA damage and may interact with base excision repair (BER) enzymes. Here, we investigated APE1 and OGG1 expression, localization and activity after oxidative stress in XPC-deficient cells. The endogenous APE1 and OGG1 mRNA levels were lower in XPC-deficient fibroblasts. However, XPC-deficient cells did not show hypersensitivity to oxidative stress compared with NER-proficient cells. To confirm the impact of an XPC deficiency in regulating APE1 and OGG1 expression and activity, we established an XPC-complemented cell line. Although the XPC complementation was only partial and transient, the transfected cells exhibited greater OGG1 expression and activity compared with XPC-deficient cells. However, the APE1 expression and activity did not significantly change. Furthermore, we observed a physical interaction between the XPC and APE1 proteins. Together, the results indicate that the responses of XPC-deficient cells under oxidative stress may not only be associated with NER deficiency per se but may also include new XPC functions in regulating BER proteins.

Φ-score: A cell-to-cell phenotypic scoring method for sensitive and selective hit discovery in cell-based assays.

Phenotypic screening monitors phenotypic changes induced by perturbations, including those generated by drugs or RNA interference. Currently-used methods for scoring screen hits have proven to be problematic, particularly when applied to physiologically relevant conditions such as low cell numbers or inefficient transfection. Here, we describe the Φ-score, which is a novel scoring method for the identification of phenotypic modifiers or hits in cell-based screens. Φ-score performance was assessed with simulations, a validation experiment and its application to gene identification in a large-scale RNAi screen. Using robust statistics and a variance model, we demonstrated that the Φ-score showed better sensitivity, selectivity and reproducibility compared to classical approaches. The improved performance of the Φ-score paves the way for cell-based screening of primary cells, which are often difficult to obtain from patients in sufficient numbers. We also describe a dedicated merging procedure to pool scores from small interfering RNAs targeting the same gene so as to provide improved visualization and hit selection.

E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells.

E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.