Project description:In order to facilitate the identification of factors and pathways in the cellular response to UV-induced DNA damage, several descriptive proteomic screens and a functional genomics screen were performed in parallel. Numerous factors could be identified with high confidence when the screen results were superimposed and interpreted together, incorporating biological knowledge. A searchable database, bioLOGIC, which provides access to relevant information about a protein or process of interest, was established to host the results and facilitate data mining. Besides uncovering roles in the DNA damage response for numerous proteins and complexes, including Integrator, Cohesin, PHF3, ASC-1, SCAF4, SCAF8, and SCAF11, we uncovered a role for the poorly studied, melanoma-associated serine/threonine kinase 19 (STK19). Besides effectively uncovering relevant factors, the multiomic approach also provides a systems-wide overview of the diverse cellular processes connected to the transcription-related DNA damage response.
Project description:UV-DDB, a key protein in human global nucleotide excision repair (NER), binds avidly to abasic sites and 8-oxo-guanine (8-oxoG), suggesting a noncanonical role in base excision repair (BER). We investigated whether UV-DDB can stimulate BER for these two common forms of DNA damage, 8-oxoG and abasic sites, which are repaired by 8-oxoguanine glycosylase (OGG1) and apurinic/apyrimidinic endonuclease (APE1), respectively. UV-DDB increased both OGG1 and APE1 strand cleavage and stimulated subsequent DNA polymerase β-gap filling activity by 30-fold. Single-molecule real-time imaging revealed that UV-DDB forms transient complexes with OGG1 or APE1, facilitating their dissociation from DNA. Furthermore, UV-DDB moves to sites of 8-oxoG repair in cells, and UV-DDB depletion sensitizes cells to oxidative DNA damage. We propose that UV-DDB is a general sensor of DNA damage in both NER and BER pathways, facilitating damage recognition in the context of chromatin.
Project description:While it is well-established that UV radiation threatens genomic integrity, the precise mechanisms by which cells orchestrate DNA damage response and repair within the context of 3D genome architecture remain unclear. Here, we address this gap by investigating the UV-induced reorganization of the 3D genome and its critical role in mediating damage response. Employing temporal maps of contact matrices and transcriptional profiles, we illustrate the immediate and holistic changes in genome architecture post-irradiation, emphasizing the significance of this reconfiguration for effective DNA repair processes. We demonstrate that UV radiation triggers a comprehensive restructuring of the 3D genome organization at all levels, including loops, topologically associating domains and compartments. Through the analysis of DNA damage and excision repair maps, we uncover a correlation between genome folding, gene regulation, damage formation probability, and repair efficacy. We show that adaptive reorganization of the 3D genome is a key mediator of the damage response, providing new insights into the complex interplay of genomic structure and cellular defense mechanisms against UV-induced damage, thereby advancing our understanding of cellular resilience.
Project description:In order to characterize the genome-wide transcriptional response of the hyperthermophilic, aerobic crenarchaeote Sulfolobus solfataricus to UV damage, we used high-density DNA microarrays which covered 3,368 genetic features encoded on the host genome, as well as the genes of several extrachromosomal genetic elements. While no significant up-regulation of genes potentially involved in direct DNA damage reversal was observed, a specific transcriptional UV response involving 55 genes could be dissected. Although flow cytometry showed only modest perturbation of the cell cycle, strong modulation of the transcript levels of the Cdc6 replication initiator genes was observed. Up-regulation of an operon encoding Mre11 and Rad50 homologs pointed to induction of recombinational repair. Consistent with this, DNA double-strand breaks were observed between 2 and 8 h after UV treatment, possibly resulting from replication fork collapse at damaged DNA sites. The strong transcriptional induction of genes which potentially encode functions for pilus formation suggested that conjugational activity might lead to enhanced exchange of genetic material. In support of this, a statistical microscopic analysis demonstrated that large cell aggregates formed upon UV exposure. Together, this provided supporting evidence to a link between recombinational repair and conjugation events.
Project description:DNA damage provokes DNA repair, cell-cycle regulation and apoptosis. This DNA-damage response encompasses gene-expression regulation at the transcriptional and post-translational levels. We show that cellular responses to UV-induced DNA damage are also regulated at the post-transcriptional level by microRNAs. Survival and checkpoint response after UV damage was severely reduced on microRNA-mediated gene-silencing inhibition by knocking down essential components of the microRNA-processing pathway (Dicer and Ago2). UV damage triggered a cell-cycle-dependent relocalization of Ago2 into stress granules and various microRNA-expression changes. Ago2 relocalization required CDK activity, but was independent of ATM/ATR checkpoint signalling, whereas UV-responsive microRNA expression was only partially ATM/ATR independent. Both microRNA-expression changes and stress-granule formation were most pronounced within the first hours after genotoxic stress, suggesting that microRNA-mediated gene regulation operates earlier than most transcriptional responses. The functionality of the microRNA response is illustrated by the UV-inducible miR-16 that downregulates checkpoint-gene CDC25a and regulates cell proliferation. We conclude that microRNA-mediated gene regulation adds a new dimension to the DNA-damage response.
Project description:Genetic screens in Saccharomyces cerevisiae have allowed for the identification of many genes as sensors or effectors of DNA damage, typically by comparing the fitness of genetic mutants in the presence or absence of DNA-damaging treatments. However, these static screens overlook the dynamic nature of DNA damage response pathways, missing time-dependent or transient effects. Here, we examine gene dependencies in the dynamic response to ultraviolet radiation-induced DNA damage by integrating ultra-high-density arrays of 6144 diploid gene deletion mutants with high-frequency time-lapse imaging. We identify 494 ultraviolet radiation response genes which, in addition to recovering molecular pathways and protein complexes previously annotated to DNA damage repair, include components of the CCR4-NOT complex, tRNA wobble modification, autophagy, and, most unexpectedly, 153 nuclear-encoded mitochondrial genes. Notably, mitochondria-deficient strains present time-dependent insensitivity to ultraviolet radiation, posing impaired mitochondrial function as a protective factor in the ultraviolet radiation response.
Project description:Loss of function of KIND1, a cytoskeletal protein involved in β1-integrin function, causes Kindler syndrome, a genetic disease characterized by skin fragility, photosensitivity, and increased risk of squamous cell carcinoma. Dysregulation of β1-integrin underlies Kindler syndrome skin fragility. However, the mechanisms underlying squamous cell carcinoma susceptibility are unclear. Here, we demonstrate that gene silencing of KIND1 decreased keratinocyte proliferation and increased apoptosis in vitro and in skin grafts regenerated on mice, which was correlated with reduced cyclinB1. In addition, KIND1 loss sensitized keratinocytes to cytokine and UV-induced NF-κB and c-Jun N-terminal kinase activation and upregulation of CXCL10 and tumor necrosis factor-α. Moreover, KIND1 loss impaired DNA repair, as indicated by the increased detection of γH2AX and cyclobutane pyrimidine dimers 24 hours after UVB radiation. Genetic or pharmacological c-Jun N-terminal kinase inhibition and NF-κB inhibition markedly reduced cyclobutane pyrimidine dimers-positive cells. Further, we show that KIND1 was regulated by JunB at the transcriptional level and, like JunB, it was downregulated in human squamous cell carcinoma cells. Together, these results indicate that KIND1 is important not only for keratinocyte proliferation but also for the suppression of UV-induced inflammation and DNA damage. These latter findings support a tumor suppressor function for KIND1, and identify c-Jun N-terminal kinase and NF-κB as potential therapeutic targets for prevention of squamous cell carcinoma in patients with Kindler syndrome.
Project description:PrimPol is a recently identified member of the archaeo-eukaryote primase (AEP) family of primase-polymerases. It has been shown that this mitochondrial and nuclear localized enzyme plays roles in the maintenance of both unperturbed replication fork progression and in the bypass of lesions after DNA damage. Here, we utilized an avian (DT40) knockout cell line to further study the consequences of loss of PrimPol (PrimPol(-/-)) on the downstream maintenance of cells after UV damage. We report that PrimPol(-/-) cells are more sensitive to UV-C irradiation in colony survival assays than Pol η-deficient cells. Although this increased UV sensitivity is not evident in cell viability assays, we show that this discrepancy is due to an enhanced checkpoint arrest after UV-C damage in the absence of PrimPol. PrimPol(-/-) arrested cells become stalled in G2, where they are protected from UV-induced cell death. Despite lacking an enzyme required for the bypass and maintenance of replication fork progression in the presence of UV damage, we show that PrimPol(-/-) cells actually have an advantage in the presence of a Chk1 inhibitor due to their slow progression through S-phase.
Project description:Regulation of p53 by ubiquitination and deubiquitination is important for its function. In this study, we demonstrate that USP24 deubiquitinates p53 in human cells. Functional USP24 is required for p53 stabilization, and p53 destabilization in USP24-depleted cells can be corrected by the forced expression of USP24. We show that USP24 depletion renders cells resistant to apoptosis after UV irradiation, consistent with the requirement of USP24 for p53 stabilization and PUMA activation in vivo. Additionally, purified USP24 protein is able to cleave ubiquitinated p53 in vitro. Importantly, cells with USP24 depletion exhibited significantly elevated mutation rates at the endogenous HPRT locus, implying an important role for USP24 in maintaining genome stability. Our data reveal that the USP24 deubiquitinase regulates the DNA damage response by directly targeting the p53 tumor suppressor.