Proton therapy is increasingly used, due to its favourable physical properties such as the Bragg peak, which enable precise dose deposition while minimizing exposure to surrounding healthy tissues. One of the main indications for proton therapy is head and neck tumours. In addition to their therapeutic relevance, protons are also a key component of space radiation spectrum, raising concerns about their biological impact on normal tissues, particularly the central nervous system. Despite this growing concern, the effects of proton irradiation on adult post-mitotic neurons in vivo remain poorly understood. In this context, we analysed the impact of proton central nervous system micro-irradiation using the MIRCOM microbeam, in young adults of Caenorhabditis elegans, with a focus on DNA damage and behaviour. Protons induce localized oxidative DNA damage, confirmed by an increase in 8-oxoG signals within both the nuclei and cytoplasm of irradiated neurons. The biological response remained strictly confined to the targeted area; no signal propagation or damage was observed in neighbouring non-irradiated tissues. Despite molecular changes, there was no observable impact on behaviours such as motor activity, foraging, habituation, or touch-avoidance. These results suggest that proton exposure in post-mitotic neurons induces complex oxidative DNA damage and activates specific BER pathways without compromising functional behaviour or triggering systemic bystander effects. These insights are important for refining clinical radiation protection and understanding risks in space exploration.
Oxidative stress is widely implicated in aging and chronic disease, but current insights have not led to the definition of a lesion state in the progression of disease or to broad guidance of therapy. So far, reactive oxygen species describe a chemical environment rather than a specific molecular entity. In contrast, 8-oxoG represents a defined oxidative DNA lesion with a defined genomic context impacting biological processes. Here, we propose that 8-oxoG and its downstream repair intermediates define a functionally relevant lesion state at telomeres. At chromosome ends, oxidized guanine can impair replication, activate DNA damage signalling, contribute to telomere attrition and drive senescence. Still, persistent lesions and the toxicity of downstream base excision repair intermediates contribute to this process. This distinction has direct therapeutic consequences: OGG1 inhibitors suppress inflammatory and transcriptional responses, whereas activators and organocatalytic switches promote lesion clearance. We propose that going forward, the choice between these strategies should depend on the dominant lesion state rather than global measures of oxidative stress.
We applied an unsupervised interactome analysis with the RAG2 C-terminal region (R2CT) in v-abl pro-B cells undergoing V(D)J recombination. Mass-spectrometry analyses showed that Ku70 and Ku80 were among the top 10 hits. To further strengthen these observations, we performed Proximity Ligation Assay (PLA) and characterize the existence of a GFP-R2CT-Ku complex formation in cellulo. The interaction of several partners with Ku70/80 (Ku) through Ku-binding motifs (KBMs) in their sequences governs their enrolment in NHEJ repair complexes. Through sequence analysis, we identified a KBM within R2CT (R-KBM, amino acids 589-527). We confirmed by calorimetry a specific micromolar interaction between this RAG2 region and Ku70/80/DNA complex. The RAG2 motif KBM can be subdivided in two conserved parts that have no interaction individually. AlphaFold2 prediction coupled with molecular dynamic simulations indicate that the C-terminal part of the RAG2 motif interacts with Ku80 on the same site than the NHEJ factor XLF. These in silico analyses indicated that the N-terminal part of the RAG2 motif interacts with DNA adjacent to Ku with a major role of the K503 residue in agreement with disruption of the interaction observed with the K503E mutant. This study further extends the large ensemble of proteins recruited at DSBs by KBM motifs and substantiates the model of a tight coupling between DNA breakage and repair during V(D)J recombination, mediated by the Ku-RAG2 C-terminus interaction.
DNA double-strand breaks (DSBs) threaten genomic integrity, with erroneous repair leading to chromosomal rearrangements and pathologies. In eukaryotes, DSBs are primarily repaired via non-homologous end-joining (NHEJ) or homologous recombination (HR). HR restores genetic information by using an undamaged homologous sequence as a template, a process dependent on Rad51-mediated homology search. This review synthesizes recent advances in our understanding of HR, with a focus on the homology search process in mitotic cells, primarily using Saccharomyces cerevisiae as a model organism. We explore factors that limit recombination efficiency and discuss how Rad51 filament dynamics overcome spatial and temporal challenges imposed by nuclear architecture and chromatin dynamics, to ensure efficient HR. Key insights include the dynamic behavior of Rad51 filaments, which undergo cycles of compaction and extension, thereby optimizing exploration of the nuclear volume and increasing the likelihood of encountering distant homologous sequences. The interplay between long-range resection, filament elongation, and nuclear constraints further shapes the search process, balancing the need for extensive exploration with the risks of excessive DNA degradation and ectopic recombination. Collectively, these findings support an integrated model in which the efficiency and accuracy of homologous recombination are governed by regulated Rad51 filament dynamics and the constraints imposed by nuclear architecture.
DNA double-strand breaks (DSBs) are the most harmful type of DNA damage. Efficient repair of DSBs is critical for cell survival and contributes to cancer resistance. Protein kinases have been found to be involved in this sophisticated process. In this study, we utilized an optimized loss-of-function screen for a customized siRNA Premix Library to identify kinases involved in HR repair. NEK3 was identified as a novel regulator required for HR repair. Furthermore, depletion of NEK3 significantly increased the sensitivity of cancer cells to ionizing radiation. Mechanistically, NEK3 promotes CtIP transcription, which contributes to HR-mediated DNA damage repair. In summary, our findings uncovered a novel function of NEK3 in DNA damage repair, suggesting a potential therapeutic strategy to overcome cancer resistance to chemotherapy and radiotherapy.
DNA base damage is the most common form of lesion, occurring approximately 10,000 times per cell a day. Such damage arises either from the incorporation of damaged nucleotides or from direct modification of bases within DNA strand. 5-hydroxymethyl-2´-deoxyuridine (5-hmdU) is a relatively common base lesion generated through the incorporation of oxidized thymidine or through the deamination of 5-hydroxymethyl-2´-deoxycytidine, a product produced during epigenetic regulatory process. However, the mechanisms underlying the repair and cellular tolerance to 5-hmdU have not been fully elucidated. Here, we explored genome maintenance factors required for cellular tolerance to 5-hmdU using a mutant cell collection deficient in individual genome maintenance factors derived from chicken DT40 cell line. We found that PARP1 is the most critical factor for cellular tolerance to 5-hmdU. PARP1-/- cells exhibited delayed replication fork progression following 5-hmdU exposure, indicating that DNA replication is acutely impaired upon 5-hmdU incorporation. Following the incorporation of 5-hmdU into nascent DNA, PARP1-deficient cells showed massive single strand breaks, which subsequently led to double strand breaks and ultimately apoptosis. Notably, these defects observed in PARP1-/- cells were completely rescued by the loss of SMUG1, a DNA glycosylase responsible for the removal of 5-hmdU from genome. These findings indicate that the SMUG1-dependent processing of 5-hmdU in nascent DNA is highly toxic in the absence of PARP1. Collectively, our results reveal a previously unappreciated role of PARP1 in counteracting toxic effects of SMUG1-mediated 5-hmdU processing during DNA replication.
Nucleotide excision repair (NER) is a principal DNA repair system that can remove a wide variety of DNA lesions caused mainly by environmental agents such as ultraviolet irradiation and chemical compounds. In global genome NER (GG-NER) of higher eukaryotes, initial lesion recognition depends on the specific DNA-binding proteins XPC and UV-DDB. Recent biochemical and structural studies have revealed the sophisticated molecular mechanism of DNA lesion recognition, which accounts for the versatility, efficiency, and accuracy of GG-NER. On the other hand, in living cells, dynamic regulation of higher-order chromatin structures is required to enable lesion recognition and subsequent repair reactions, whereas a comprehensive understanding of the underlying principles and mechanisms remains to be established. In this article, we summarize current knowledge of the mechanism and regulation of DNA lesion recognition in GG-NER, with a particular focus on the functional impacts of chromatin dynamics.
DNA double-strand breaks (DSBs) are among the most cytotoxic forms of chromosomal lesions and are primarily repaired through homologous recombination (HR) or non-homologous end joining (NHEJ). The precise repair of DSBs via HR necessitates 5'-3' end resection to generate 3'-single-stranded DNA (ssDNA) overhangs, which function as templates for repair synthesis. However, the proteins involved in HR, particularly those acting in the critical early stages preceding DNA end resection, and their regulatory mechanisms in response to ionizing radiation (IR), remain incompletely characterized. In this study, we identify the nuclear receptor co-activator NCOA4 as a novel DNA damage responsive protein. We demonstrate that this protein is recruited to sites of DNA damage and is enriched during the S/G2 phase of the cell cycle. Immunofluorescence and reporter gene assays demonstrate that depletion of NCOA4 reduces the IR-induced foci formation of RAD51 and RPA2 and impairs HR efficiency. Mechanistically, NCOA4 interacts with the AAA + ATPases RUVBL1/2, and depletion of RUVBL1/2 has been shown to reduce the recruitment of NCOA4 at DNA damage sites. The knockdown of RUVBL1 or RUVBL2 phenocopies NCOA4 deficiency, and simultaneous knockdown of RUVBL1/2 and NCOA4 does not further reduce the RPA2 RIF, confirming that the entire NCOA4-RUVBL1/2 complex acts together to promote HR. Furthermore, depletion of NCOA4 has been shown to sensitize cancer cells to radiotherapy in tumor-bearing nude mouse models. Consequently, the present findings indicate that the NCOA4-RUVBL1/2 axis is capable of recognizing DNA double-strand breaks and promoting the homologous recombination repair pathway, thereby contributing to the maintenance of genomic integrity. This process may potentially play a role in modulating radioresistance in malignant tumors and expanding the landscape of therapeutic targets.
Xeroderma pigmentosum (XP) is a rare recessive autosomal genodermatosis caused by defects in nucleotide excision repair (NER). Patients with XP are extremely sensitive to ultraviolet-light, resulting in an increased incidence of skin cancers in sun-exposed areas. Thanks to improved photoprotection and therapeutic education, the life expectancy of patients with XP has increased substantially. Recently, however, it has been observed that some XP patients are also at very high risk of developing internal tumors. Notably, 50% of these tumors correspond to severe myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML) occurring in XP-C patients younger than 25 years, almost 50 years earlier than for the general population. To explain this striking susceptibility, we hypothesize that two distinct but complementary mechanisms contribute to the high risk of hematologic malignancies in XP-C patients. First, preserved transcription-coupled repair combined with defective global-genome repair in XP-C cells, in the presence of as-yet-uncharacterized endogenous bulky DNA lesions, results in a more than 25-fold increase in mutation frequency in XP-C-associated hematologic malignancies compared with corresponding tumors in the general population. Whole-genome sequencing of XP-C hematologic malignancies reveals a COSMIC SBS8 mutational signature characteristic of NER deficiency. Second, the XPC protein has been identified as a co-factor of RNA polymerase II regulating hundreds of genes. Loss of full-length XPC leads to dysregulation of multiple gene pathways, including those involved in hematopoietic, immunologic, and oncogenic processes. We propose that together, the accumulation of driver-gene mutations and the disruption of hematopoietic regulatory pathways may account for the exceptionally high incidence and severity of MDS/AML observed in young XP-C patients.
Telomeric sequences are hotspots for ultraviolet light (UV) induced cyclobutane pyrimidine dimers (CPD) and pyrimidine(6-4)pyrimidone photoproducts (6-4 PP), due to pyrimidine runs on both the TTAGGG and CCCTAA containing strands. Photoproducts are repaired by global genome nucleotide excision repair (GG-NER) or by transcription-coupled (TC-NER) in regions of active transcription. Since telomeres are transcribed into long telomeric repeat-containing RNA (TERRA) molecules, here we tested roles for both TC-NER and GG-NER in telomere stability following UVC irradiation. XPC-deficient cells, incapable of GG-NER, failed to exhibit significant reductions in 6-4 PPs and CPDs at telomeres during recovery times, indicating that TC-NER cannot compensate for detectable photoproduct removal at telomeres when GG-NER is absent. TERRA analysis confirmed active telomere transcription in these cell lines. Loss of total NER or specifically GG-NER in XPA-deficient or XPC-deficient cells, respectively, increased telomere losses and telomere fragility following UV irradiation. These data provide direct evidence that NER is required to prevent UV damage-induced telomere aberrations and that transcription at telomeres is likely insufficient to drive substantial TC-NER-mediated photoproduct removal.
BACKGROUND/AIM:This study aimed to evaluate the anticancer efficacy of 5-Fluorouracil (5-FU) in Caco-2 human colorectal adenocarcinoma cells treated with siRNA-mediated MLH1 gene inhibition, in terms of cell viability, apoptosis, and related gene/protein expression. MATERIALS AND METHODS:Caco-2 cells were treated with a dose of 10 µM 5-FU. Cell viability was assessed by CVDK-8 analysis, and apoptosis was determined by flow cytometry using Annexin V-FITC/PI staining. Expression levels of MLH1, CDK2, CDK4, CDK5, CDK6, PTEN, EGFR, mTOR, PI3K, AKT3, ERK, PARP1, and GAPDH genes were examined by RT-qPCR analysis. Protein expression levels were analyzed using Western blot for PI3K, p-AKT, AKT1, mTOR, MAPK-p38, Caspase-3, Bcl-2, STAT3, JAK2, MLH1, MDR1, p53, and proteins; β-Actin was used as an internal control. RESULTS:CVDK-8 analysis showed that application of 10 µM 5-FU significantly reduced cell viability in Caco-2 cells. Flow cytometry results revealed a significant increase in both early- and late-apoptotic cell populations following 5-FU treatment. DISCUSSION:Gene and protein expression analyses showed significant changes in signaling pathways associated with apoptosis and cell proliferation. In this study, the effects of combining MLH1 gene silencing with 5-FU on colorectal cancer cells were comprehensively evaluated using Cell viability, RT-qPCR, flow cytometry, and Western blot analyses. It was determined that the combined application significantly reduced cell viability and increased apoptosis. Molecular-level findings on gene and protein expression support this effect. The results suggest that MLH1 gene silencing may increase sensitivity to 5-FU, offering a potential combination therapy approach in colorectal cancer.
DNA replication is a strictly regulated process during cell proliferation to ensure faithful duplication of the genome. Its firing and elongation can be arrested or temporally inhibited in response to a variety of internal and external causes. Inside cells numerous factors including cell cycle checkpoints, protein kinases, and others are involved in the control of this process to maintain genome integrity. Here, we describe that NuMA, a nuclear scaffolding protein, plays an important role in regulating DNA replication. We show that NuMA is present at active replication forks, and its deficiency impairs cell viability, reduces the replication fork speed and increases origin firing, increases the level of γH2AX and activates the ATM-CHK2 DNA damage response pathway. Mechanistically, our results show that NuMA depletion reduces the association of multiple key replisome proteins to replication forks, suggesting that NuMA promotes the association of replisome proteins to ongoing forks. Our study uncovers a novel function of NuMA in maintaining genome stability, providing new insights into the important role of nuclear structural proteins in safeguarding DNA replication.
PARP1 is a key regulator of DNA damage responses. Reports from cell and animal models suggest that when excessively active, PARP1 exacerbates oxidative stress, mitochondrial dysfunction, and neuroinflammation, which are indicative of Alzheimer's disease (AD) pathogenesis. Recently, there have been great advances in the biology of PARP and modulation of PARP for potential repurposing for neuroprotection. This narrative review details the molecular and interrelationship of PARP1 activity and Alzheimer's progression while trying to implement strategies to mitigate PARP1 activity. This includes molecular pathways, gene associations, and increasing interest in nicotinamide therapies and next-generation PARP-inhibitory drugs. It has been shown that uncontrolled regulation of PARP1 activity will lead to cell death via parthanatos. Increased destruction of cellular NAD+ and ATP, loss of mitochondrial energy, and the unbalanced, unregulated NF-κB neuroinflammation will focus on cell death. Preclinical studies have shown that nicotinamide and NAD+ , cell-permeable PARP1-active agents, enhance mitochondrial function and cognitive resilience. Moreover, brain-penetrant inhibitors such as veliparib and AZD9574 offer enhanced selectivity and access to the central nervous system (CNS). The future calls for CNS-optimized, selective inhibitors that combine safety with bioavailability. This review highlights the exciting possibilities of PARP1 modulation not only for symptomatic relief but also as a marked improvement for the future of treating AD.
Uracil arises in DNA either by spontaneous deamination of cytosine or misincorporation of dUMP by DNA polymerases. Cytosine deamination (to uracil), if unrepaired, may lead to GC to AT mutation. Uracil DNA glycosylases (UDGs) initiate the base excision repair (BER) pathway by excising uracil from DNA. Here, we report a novel uracil DNA glycosylase, CC2084, UdgC, from Caulobacter crescentus, an α-proteobacterium. Despite lacking significant overall sequence similarity with the known UDGs, CC2084 contains motif A (GQAPG) resembling to that of the family I UDGs (Ung), and motif B (HPSWRNT) similar to that of the family V UDGs (UdgB). In addition to uracil, CC2084 removes hypoxanthine and xanthine from single-, and double-, stranded DNAs; and ethenoadenine from double stranded DNA. Also, we notice that CC2084 activity is inhibited by the reaction products, uracil and AP site containing DNA, as well as by the phage-encoded Ugi, which was known to target only the family I UDGs. CC2084 rescues the mutator phenotype of E. coli ∆ung, an effect that is attenuated when co-expressed with Ugi. Phylogenetically, CC2084 forms a separate group of UDGs. The presence of CC2084 in the highly GC-rich genome of Caulobacter may compensate for the absence of the highly efficient Ung protein.
A range of genome maintenance factors respond to endogenous and exogenous DNA damage to prevent mutations and cell death. The scaffold protein, Rtt107, is important for the growth of cells exposed to DNA-damaging agents in the budding yeast Saccharomyces cerevisiae. Rtt107 binds to a diverse array of partner proteins and responds to DNA damage by localizing to phosphorylated histone H2A. Rad55-Rad57, a heterodimer involved in DNA repair, also binds to Rtt107, but the function of the Rtt107-Rad55-Rad57 complex remains unclear. In addition to their sensitivity to DNA-damaging agents, rtt107∆ mutants exhibit spontaneous genome instability phenotypes, including spontaneous loss of heterozygosity (LOH) caused by crossovers and other genetic events. However, the binding partners with which Rtt107 interacts to prevent spontaneous genome instability have yet to be elucidated. Here, we showed that Rtt107 acts in the same pathway as Rad55 to limit LOH, specifically by preventing crossover events. A rad55-S404A mutation largely disrupted the interaction between Rtt107 and Rad55-Rad57, resulting in increased LOH and crossover rates, consistent with the contribution of Rtt107-Rad55-Rad57 interaction to genome stability. Strikingly, an rtt107-K887M mutation that reduces Rtt107 recruitment to H2A did not result in an LOH phenotype, suggesting that the role of Rtt107 in preventing LOH is distinct from its function as an H2A-binding scaffold. Taken together, our observations suggested that Rtt107 limits spontaneous LOH and crossover events in part by binding to Rad55 in a manner dependent on Rad55-S404.
Brh2, the streamlined BRCA2 family protein from Ustilago maydis, directs homologous recombination by modulating Rad51 filament dynamics through a single BRC element. Here, we describe an unusual antimorphic mutation within the BRC β-hairpin of Brh2, in which the pivotal phenylalanine residue in the sequence mimicking the Rad51 polymerization motif is replaced by methionine. This methionine-for-phenylalanine substitution in the BRC of Brh2 results in a DNA repair defect more severe than the null allele, while the cognate methionine-for-phenylalanine substitution in Rad51's polymerization motif supports normal function. The dichotomy suggests that the switch to methionine from phenylalanine exerts distinct structural effects depending on molecular context. Suppression of the antimorphic phenotype by a secondary mutation (T296A) destabilizing the β-hairpin implies that the deleterious activity of the mutant variant requires an intact BRC fold. Relocation of the mutant BRC to an ectopic site within Brh2 mitigates the defect. This indicates there is positional dependence and hierarchical use of BRC modules within the protein. Biochemical assays with BRC polypeptides demonstrate that the BRCF294M variant is reduced in ability to form stable complexes with free Rad51. However, analysis shows BRCF294M engages Rad51-ssDNA filaments aberrantly promoting atypical aggregation rather than normal filament dynamics. These findings suggest a mechanistic basis for this antimorphic BRC mutation and highlight the critical influence of BRC structural context in governing Rad51 filament dynamics and DNA repair proficiency.
