RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid–liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, and 53BP1 and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSB repair complexes.

Histone H2AX phosphorylation is an early signalling event triggered by DNA double-strand breaks (DSBs). To elucidate the elementary units of phospho-H2AX-labelled chromatin, we integrate super-resolution microscopy of phospho-H2AX during DNA repair in human cells with genome-wide sequencing analyses. Here we identify phospho-H2AX chromatin domains in the nanometre range with median length of ∼75 kb. Correlation analysis with over 60 genomic features shows a time-dependent euchromatin-to-heterochromatin repair trend. After X-ray or CRISPR-Cas9-mediated DSBs, phospho-H2AX-labelled heterochromatin exhibits DNA decondensation while retaining heterochromatic histone marks, indicating that chromatin structural and molecular determinants are uncoupled during repair. The phospho-H2AX nano-domains arrange into higher-order clustered structures of discontinuously phosphorylated chromatin, flanked by CTCF. CTCF knockdown impairs spreading of the phosphorylation throughout the 3D-looped nano-domains. Co-staining of phospho-H2AX with phospho-Ku70 and TUNEL reveals that clusters rather than nano-foci represent single DSBs. Hence, each chromatin loop is a nano-focus, whose clusters correspond to previously known phospho-H2AX foci.

Objectives The HIV restriction factor, SAMHD1 (SAM domain and HD domain-containing protein 1), is a triphosphohydrolase that degrades deoxyribonucleoside triphosphates (dNTPs). Mutations in SAMHD1 cause Aicardi–Goutières syndrome (AGS), an inflammatory disorder that shares phenotypic similarity with systemic lupus erythematosus, including activation of antiviral type 1 interferon (IFN). To further define the pathomechanisms underlying autoimmunity in AGS due to SAMHD1 mutations, we investigated the physiological properties of SAMHD1. Methods Primary patient fibroblasts were examined for dNTP levels, proliferation, senescence, cell cycle progression and DNA damage. Genome-wide transcriptional profiles were generated by RNA sequencing. Interaction of SAMHD1 with cyclin A was assessed by coimmunoprecipitation and fluorescence cross-correlation spectroscopy. Cell cycle-dependent phosphorylation of SAMHD1 was examined in synchronised HeLa cells and using recombinant SAMHD1. SAMHD1 was knocked down by RNA interference. Results We show that increased dNTP pools due to SAMHD1 deficiency cause genome instability in fibroblasts of patients with AGS. Constitutive DNA damage signalling is associated with cell cycle delay, cellular senescence, and upregulation of IFN-stimulated genes. SAMHD1 is phosphorylated by cyclin A/cyclin-dependent kinase 1 in a cell cycle-dependent manner, and its level fluctuates during the cell cycle, with the lowest levels observed in G₁/S phase. Knockdown of SAMHD1 by RNA interference recapitulates activation of DNA damage signalling and type 1 IFN activation. Conclusions SAMHD1 is required for genome integrity by maintaining balanced dNTP pools. dNTP imbalances due to SAMHD1 deficiency cause DNA damage, leading to intrinsic activation of IFN signalling. These findings establish a novel link between DNA damage signalling and innate immune activation in the pathogenesis of autoimmunity.

Immune recognition of cytosolic DNA represents a central antiviral defence mechanism. Within the host, short single-stranded DNA (ssDNA) continuously arises during the repair of DNA damage induced by endogenous and environmental genotoxic stress. Here we show that short ssDNA traverses the nuclear membrane, but is drawn into the nucleus by binding to the DNA replication and repair factors RPA and Rad51. Knockdown of RPA and Rad51 enhances cytosolic leakage of ssDNA resulting in cGAS-dependent type I IFN activation. Mutations in the exonuclease TREX1 cause type I IFN-dependent autoinflammation and autoimmunity. We demonstrate that TREX1 is anchored within the outer nuclear membrane to ensure immediate degradation of ssDNA leaking into the cytosol. In TREX1-deficient fibroblasts, accumulating ssDNA causes exhaustion of RPA and Rad51 resulting in replication stress and activation of p53 and type I IFN. Thus, the ssDNA-binding capacity of RPA and Rad51 constitutes a cell intrinsic mechanism to protect the cytosol from self DNA.

UVA (320–400 nm) represents the main spectral component of solar UV radiation, induces pre-mutagenic DNA lesions and is classified as Class I carcinogen. Recently, discussion arose whether UVA induces DNA double-strand breaks (dsbs). Only few reports link the induction of dsbs to UVA exposure and the underlying mechanisms are poorly understood. Using the Comet-assay and γH2AX as markers for dsb formation, we demonstrate the dose-dependent dsb induction by UVA in G ₁ -synchronized human keratinocytes (HaCaT) and primary human skin fibroblasts. The number of γH2AX foci increases when a UVA dose is applied in fractions (split dose), with a 2-h recovery period between fractions. The presence of the anti-oxidant Naringin reduces dsb formation significantly. Using an FPG-modified Comet-assay as well as warm and cold repair incubation, we show that dsbs arise partially during repair of bi-stranded, oxidative, clustered DNA lesions. We also demonstrate that on stretched chromatin fibres, 8-oxo-G and abasic sites occur in clusters. This suggests a replication-independent formation of UVA-induced dsbs through clustered single-strand breaks via locally generated reactive oxygen species. Since UVA is the main component of solar UV exposure and is used for artificial UV exposure, our results shine new light on the aetiology of skin cancer.

The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.