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.

Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA–DNA hybrid with a displaced non-template DNA strand. We developed RNA–DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DDX41 in opposing R-loop and double strand DNA break accumulation in promoters. DDX41 is enriched in promoter regions in vivo, and can unwind RNA–DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied with replication stress, an increase in the formation of double strand DNA breaks and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41.

Circadian rhythms are driven by molecular clocks composed of interlocking transcription/translation feedback loops. CRYPTOCHROME (CRY) proteins are critical components of these clocks and repress the activity of the transcription factor heterodimer CLOCK/BMAL1. Unlike the homologous DNA repair enzyme 6-4 PHOTOLYASE, CRYs have extended carboxyl-terminal tails and cannot repair DNA damage (reviewed in ). Unlike mammals, Xenopus laevis contains both CRYs (xCRYs) and 6-4 PHOTOLYASE (xPHOTOLYASE), providing an excellent comparative tool to study CRY repressive function. We can extend findings to CRYs in general because xCRYs share high sequence homology with mammalian CRYs. We show here that deletion of xCRYs' C-terminal domain produces proteins that are, like xPHOTOLYASE, unable to suppress CLOCK/BMAL1 activation. However, these truncations also cause the proteins to be cytoplasmically localized. A heterologous nuclear localization signal (NLS) restores the truncation mutants' nuclear localization and repressive activity. Our results demonstrate that the CRYs' C termini are essential for nuclear localization but not necessary for the suppression of CLOCK/BMAL1 activation; this finding indicates that these two functions reside in separable domains. Furthermore, the functional differences between CRYs and PHOTOLYASE can be attributed to the few amino acid changes in the conserved portions of these proteins.

Circadian rhythms control the temporal arrangement of molecular, physiological, and behavioral processes within an organism and also synchronize these processes with the external environment. A cell autonomous molecular oscillator, consisting of interlocking transcriptional/translational feedback loops, drives the approximately 24-hour duration of these rhythms. The cryptochrome protein (CRY) plays a central part in the negative feedback loop of the molecular clock by translocating to the nucleus and repressing CLOCK and BMAL1, two transcription factors that comprise the positive elements in this cycle. In order to gain insight into the inner workings of this feedback loop, we investigated the structure/ function relationships of Xenopus laevis CRY1 (xCRY1) and xCRY2 in cultured cells. The C-terminal tails of both xCRY1 and xCRY2 are sufficient for their nuclear localization but achieve it by different mechanisms. Through the generation and characterization of xCRY/photolyase chimeras, we found that the second half of the photolyase homology region (PHR) of CRY is important for repression through facilitating interaction with BMAL1. Characterization of these functional domains in CRYs will help us to better understand the mechanism of the known roles of CRYs and to elucidate new intricacies of the molecular clock.Organisms ranging from cyanobacteria to humans exhibit circadian rhythms in many processes, from gene expression to cell physiology and from hormone levels to locomotor activity. Circadian rhythms are approximately 24 hours in duration and persist in constant conditions. These oscillations do not accelerate or decelerate within a physiological range of temperatures and, importantly, can be reset by cues from the environment. Having an internal timekeeping mechanism allows an organism to temporally arrange physiological processes internally and also to anticipate changes in the external environment (reviewed in reference 1).The clocks driving these rhythms are intracellular mechanisms composed of interlocking transcriptional/translational feedback loops (reviewed in reference 23). At the core of the vertebrate molecular oscillator is a negative feedback loop that is necessary for rhythmicity (19). Two positive elements, CLOCK and BMAL1, which are basic helix-loop-helix PAS transcription factors, heterodimerize and bind to E-box enhancer elements in the promoters of the Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes, activating their transcription. The Per and Cry mRNAs are then translated, and the proteins accumulate in the cytoplasm. PER, CRY, and casein kinase Iε (CKIε) proteins form a complex in the cytoplasm, which translocates into the nucleus, where it represses CLOCK-BMAL1-mediated transcription. The repression complex is eventually degraded or dismantled, CLOCK-BMAL1 transcription is activated, and the cycle begins again (reviewed in reference 1).Since the molecular clock is temporally precise and longer in duration than most intracellular feedback loops, many regulatory str...

RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). Implicated in RNA metabolism and DNA repair, the RBP Fused-in-sarcoma (FUS) contains a prion-like domain (PLD) and undergoes reversible phase separation. Here, we report that liquid-liquid phase separation (LLPS) occurs at DNA damage foci and is necessary for the efficient recruitment of key DDR factors. We show that FUS co-purifies with the DDR factor KU and with SFPQ, another PLD-containing RBP implicated in DNA repair.Moreover, we demonstrate that FUS is required for 53BP1 localisation to DNA damage foci and for the correct recruitment of KU80 and NBS1 to sites of DNA damage. LLPS-deficient FUS variants impair retention of the DNA damage sensor KU at sites of DNA damage, and the recruitment of SFPQ. These findings provide a mechanistic function for FUS-dependent LLPS in the activation of the DDR and in the recruitment of DDR factors and RBPs at sites of DNA damage.