Background: FUS has been implicated in the DNA damage response; however, the mechanisms are unknown. Results: FUS recruitment to DNA lesions is PARP-dependent. Depletion of FUS disrupts DNA repair. Conclusion: FUS functions downstream of PARP and promotes double-strand break repair. Significance: This work identifies FUS as a novel factor at DNA lesions and furthers our understanding of RNA-binding proteins in maintaining genomic stability.

The cyclic AMP-response element-binding protein (CREB) is a bZIP family transcription factor implicated as an oncoprotein and neuron survival factor. CREB is activated in response to cellular stimuli, including cAMP and Ca 2؉ , via phosphorylation of Ser-133, which promotes interaction between the kinase-inducible domain (KID) of CREB and the KID-interacting domain of CREB-binding protein (CBP). We previously demonstrated that the interaction between CREB and CBP is inhibited by DNA-damaging stimuli through a mechanism whereby CREB is phosphorylated by the ataxia telangiectasia-mutated (ATM) protein kinase. We now show that the ATM phosphorylation sites in CREB are functionally intertwined with a cluster of coregulated casein kinase (CK) sites. We demonstrate that DNA damage-induced phosphorylation of CREB occurs in three steps. The initial event in the CREB phosphorylation cascade is the phosphorylation of Ser-111, which is carried out by CK1 and CK2 under basal conditions and by ATM in response to ionizing radiation. The phosphorylation of Ser-111 triggers the CK2-dependent phosphorylation of Ser-108 and the CK1-dependent phosphorylation of Ser-114 and Ser-117. The phosphorylation of Ser-114 and Ser-117 by CK1 then renders CREB permissive for ATM-dependent phosphorylation on Ser-121. Mutation of Ser-121 alone abrogates ionizing radiation-dependent repression of CREB-CBP complexes, which can be recapitulated using a CK1 inhibitor. Our findings outline a complex mechanism of CREB phosphorylation in which coregulated ATM and CK sites control CREB transactivation potential by modulating its CBP-binding affinity. The coregulated ATM and CK sites identified in CREB may constitute a signaling motif that is common to other DNA damage-regulated substrates.The cAMP response element-binding protein (CREB) 2 is a phosphorylation-dependent transcription factor that plays key roles in cell proliferation, homeostasis, and survival (1). Consistent with an important role of this protein in transcriptional control, a recent whole-genome chromatin immunoprecipitation procedure identified ϳ6000 different genomic loci that are occupied by CREB in vivo (2). In silico approaches have similarly identified thousands of candidate CREB target genes (3). These include a large number of genes with annotated functions in the nervous system, which is consistent with critical roles for CREB in memory, circadian rhythm entrainment, and neuron survival (4). The current compendium of CREB target genes also includes a significant number of anti-apoptosis factors, cell cycle regulators, and DNA repair enzymes, suggestive of a role for CREB in the response to DNA damage (2, 3). This role, however, remains uncharacterized.The transactivation potential of CREB is enhanced by stimuli, including cAMP and Ca 2ϩ that induce its phosphorylation on Ser-133, which lies within a phosphorylation site-rich region spanning amino acids 100 -160 called the kinase-inducible domain (KID) (1). Phosphorylation of Ser-133 promotes an interaction between the KID and...

The functionally related ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases are critical regulators of DNA damage responses in mammalian cells. ATM and ATR share highly overlapping substrate specificities and show a strong preference for the phosphorylation of Ser or Thr residues followed by Gln. In this report we used a polyreactive phosphospecific antibody (␣-pDSQ) that recognizes a subset of phosphorylated AspSer-Gln sequences to purify candidate ATM/ATR substrates. This led to the identification of phosphorylation sites in the carboxyl terminus of the minichromosome maintenance protein 3 (MCM3), a component of the hexameric MCM DNA helicase. We show that the ␣-DSQ antibody recognizes tandem DSQ phosphorylation sites (Ser-725 and Ser-732) in the carboxyl terminus of murine MCM3 (mMCM3) and that ATM phosphorylates both sites in vitro. ATM phosphorylated the carboxyl termini of mMCM3 and human MCM3 in vivo and the phosphorylated form of MCM3 retained association with the canonical MCM complex. Although DNA damage did not affect steady-state levels of chromatin-bound MCM3, the ATM-phosphorylated form of MCM3 was preferentially localized to the soluble, nucleoplasmic fraction. This finding suggests that the carboxyl terminus of chromatin-loaded MCM3 may be sequestered from ATM-dependent checkpoint signals. Finally, we show that ATM and ATR jointly contribute to UV lightinduced MCM3 phosphorylation, but that ATM is the predominant UV-activated MCM3 kinase in vivo. The carboxyl-terminal ATM phosphorylation sites are conserved in vertebrate MCM3 orthologs suggesting that this motif may serve important regulatory functions in response to DNA damage. Our findings also suggest that DSQ motifs are common phosphoacceptor motifs for ATM family kinases. The ataxia telangiectasia-mutated (ATM)2 protein kinase is a broad regulator of cellular responses to DNA damage in mammals. Mutations in ATM cause ataxia telangiectasia (A-T), a syndrome characterized by progressive cerebellar degeneration, immune defects, and cancer susceptibility (1). ATM-deficient cells are hypersensitive to ionizing radiation (IR) and radiomimetic agents and exhibit cell cycle checkpoint abnormalities and subtle DNA repair defects. These combined defects in DNA damage signaling and repair are responsible for the 100-fold increased cancer risk associated with A-T, and most likely contribute to the neuropathologic abnormalities associated with this disease.ATM belongs to the extended family of highly conserved phosphoinositide 3-kinase-related kinases (2). Within the mammalian phosphoinositide 3-kinase-related kinase family, ATM is structurally and functionally most closely related to ATR (ATM-Rad3-related) (2). ATM and ATR exhibit similar substrate specificities in vitro, and display a strong preference for the phosphorylation of substrates on Ser/Thr-Gln ((S/T)-Q) motifs. Other (S/T)-Q-directed phosphoinositide 3-kinase-related kinases include the DNA-dependent protein kinase, which mediates DNA repair by non-homologous end ...

Mutations in ATM (Ataxia telangiectasia mutated) result in Ataxia telangiectasia (A-T), a disorder[Keywords: Drosophila; neurodegeneration; Ataxia telangiectasia; ATM; cell cycle; HDAC] Supplemental material is available at http://www.genesdev.org.

cAMP response element binding protein (CREB) is a key regulator of glucose metabolism and synaptic plasticity that is canonically regulated through recruitment of transcriptional coactivators. Here we show that phosphorylation of CREB on a conserved cluster of Ser residues (the ATM/CK cluster) by the DNA damage-activated protein kinase ataxia-telangiectasia-mutated (ATM) and casein kinase1 (CK1) and casein kinase2 (CK2) positively and negatively regulates CREB-mediated transcription in a signal dependent manner. In response to genotoxic stress, phosphorylation of the ATM/CK cluster inhibited CREB-mediated gene expression, DNA binding activity and chromatin occupancy proportional to the number of modified Ser residues. Paradoxically, substoichiometric, ATM-independent, phosphorylation of the ATM/CK cluster potentiated bursts in CREB-mediated transcription by promoting recruitment of the CREB coactivator, cAMP-regulated transcriptional coactivators (CRTC2). Livers from mice expressing a non-phosphorylatable CREB allele failed to attenuate gluconeogenic genes in response to DNA damage or fully activate the same genes in response to glucagon. We propose that phosphorylation-dependent regulation of DNA binding activity evolved as a tunable mechanism to control CREB transcriptional output and promote metabolic homeostasis in response to rapidly changing environmental conditions.