DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 A. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress.

53BP1 is a p53 binding protein of unknown function that binds to the central DNA-binding domain of p53.It relocates to the sites of DNA strand breaks in response to DNA damage and is a putative substrate of the ataxia telangiectasia-mutated (ATM) kinase. To study the biological role of 53BP1, we disrupted the 53BP1 gene in the mouse. We show that, similar to ATM ؊/؊ mice, 53BP1-deficient mice were growth retarded, immune deficient, radiation sensitive, and cancer prone. 53BP1؊/؊ cells show a slight S-phase checkpoint defect and prolonged G 2 /M arrest after treatment with ionizing radiation. Moreover, 53BP1؊/؊ cells feature a defective DNA damage response with impaired Chk2 activation. These data indicate that 53BP1 acts downstream of ATM and upstream of Chk2 in the DNA damage response pathway and is involved in tumor suppression.Defects in DNA damage recognition and repair mechanisms are associated with cancer predisposition. The tumor suppressor protein p53, a sequence specific transcription factor, plays a central role in the response of mammalian cells to genotoxic stress. 53BP1 (p53 binding protein 1) was cloned as a protein that interacts with the DNA-binding domain of p53 (13). It contains a tandem BRCT (BRCA1 C terminus) motif (5) with sequence homology to the tumor suppressor BRCA1 and DNA damage checkpoint protein scRad9. 53BP1 binds through the first of its C-terminal BRCT repeats and the inter-BRCT linker region to the central DNA-binding domain of p53 (7,15) and has been shown to enhance p53-mediated transcription of reporter genes (14). More recently, in vitro studies suggest that 53BP1 participates in the cellular response to DNA damage. 53BP1 relocates to multiple nuclear foci within minutes after exposure of cells to ionizing radiation (IR) (2,22,23,28). These foci colocalize with known DNA damage response proteins such as phosphorylated H2AX, Rad50/ Mre11/NBS1, BRCA1, and Rad51 at sites of DNA lesions (2,22,23). 53BP1 becomes hyperphosphorylated in response to IR, and several lines of evidence suggest that 53BP1 is a downstream target of the ataxia telangiectasia-mutated (ATM) kinase, the product of the gene mutated in ataxia telangiectasia (2,22,28). Furthermore, 53BP1 localizes to kinetochores in mitotic cells, suggesting a potential function of 53BP1 in mitotic checkpoint signaling (16).To study the biological function of 53BP1 in mammals, we created 53BP1-deficient mice. We report here that mice lacking 53BP1 are viable and display a phenotype that partially overlaps with that of ATM-deficient mice. 53BP1-deficient mice are growth retarded, immune deficient, radiation sensitive, and cancer prone. Thus, 53BP1 is required for an appropriate cellular response to DNA damage in vivo. MATERIALS AND METHODSGene targeting and generation of 53BP1-deficient mice. A mouse 53BP1 cDNA fragment was used as a probe to isolate 53BP1 mouse genomic DNA from a mouse 129 genomic DNA phage library (Stratagene). The genomic DNA was cloned into pZErO-2 (Invitrogen), and the exon-intron structure characterized by ...

53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for “classic” nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.

53BP1 participates in the cellular response to DNA damage. Like many proteins involved in the DNA damage response, 53BP1 becomes hyperphosphorylated after radiation and colocalizes with phosphorylated H2AX in megabase regions surrounding the sites of DNA strand breaks. However, it is not yet clear whether the phosphorylation status of 53BP1 determines its localization or vice versa. In this study we mapped a region upstream of the 53BP1 C terminus that is required and sufficient for the recruitment of 53BP1 to these DNA break areas. In vitro assays revealed that this region binds to phosphorylated but not unphosphorylated H2AX. Moreover, using H2AX-deficient cells reconstituted with wild-type or a phosphorylation-deficient mutant of H2AX, we have shown that phosphorylation of H2AX at serine 140 is critical for efficient 53BP1 foci formation, implying that a direct interaction between 53BP1 and phosphorylated H2AX is required for the accumulation of 53BP1 at DNA break sites. On the other hand, radiation-induced phosphorylation of the 53BP1 N terminus by the ATM (ataxia-telangiectasia mutated) kinase is not essential for 53BP1 foci formation and takes place independently of 53BP1 redistribution. Thus, these two damage-induced events, hyperphosphorylation and relocalization of 53BP1, occur independently in the cell.The DNA of eukaryotic cells is constantly exposed to endogenous and exogenous DNA-damaging agents. To prevent the accumulation of genomic damage and avert cellular dysfunction, cells have evolved complex response mechanisms. 53BP1 was initially identified as a protein that binds to the central DNA binding domain of p53 and enhances p53-mediated transcriptional activation (1, 2). In response to genotoxic stress, 53BP1 rapidly redistributes from a diffuse nuclear localization into distinct nuclear foci suggesting that 53BP1 is involved in the DNA damage response (3-6). Moreover the C terminus of 53BP1 contains two BRCT domains, a motif found in a number of proteins implicated in various aspects of cell cycle control, recombination, and DNA repair (7,8). Subsequent studies have shown that 53BP1 becomes hyperphosphorylated in response to ionizing radiation (IR) 1 and colocalizes with phosphorylated histone H2AX (␥-H2AX) at the sites of DNA lesions (3, 4). Other proteins known to be involved in the DNA damage signaling pathway (i.e. BRCA1, Rad51, NBS1, and TopoBP1) were also found to colocalize with 53BP1 in these inducible foci (3,4,6,9). Direct evidence for an important role of 53BP1 in the DNA damage response came recently from studies using 53BP1-deficient cells. Human cell lines treated with specific small interfering RNA to silence 53BP1 expression exhibited a defect in the intra-S phase checkpoint and, at low IR doses, a partial defect in the G 2 -M checkpoint (10 -12). Moreover 53BP1-deficient mice are hypersensitive to ionizing radiation and show an increased incidence of developing thymic lymphomas (13). Several lines of evidence suggest that 53BP1 is a substrate of ATM, the kinase mutated in the hu...

Small intestine neuroendocrine tumors (SI-NETs) are the most common malignancy of the small bowel. Several clinical trials target PI3K/Akt/mTOR signaling; however, it is unknown whether these or other genes are genetically altered in these tumors. To address the underlying genetics, we analyzed 48 SI-NETs by massively parallel exome sequencing. We detected an average of 0.1 somatic single nucleotide variants (SNVs) per 10 6 nucleotides (range, 0-0.59), mostly transitions (C>T and A>G), which suggests that SI-NETs are stable cancers. 197 protein-altering somatic SNVs affected a preponderance of cancer genes, including FGFR2, MEN1, HOOK3, EZH2, MLF1, CARD11, VHL, NONO, and SMAD1. Integrative analysis of SNVs and somatic copy number variations identified recurrently altered mechanisms of carcinogenesis: chromatin remodeling, DNA damage, apoptosis, RAS signaling, and axon guidance. Candidate therapeutically relevant alterations were found in 35 patients, including SRC, SMAD family genes, AURKA, EGFR, HSP90, and PDGFR. Mutually exclusive amplification of AKT1 or AKT2 was the most common event in the 16 patients with alterations of PI3K/Akt/ mTOR signaling. We conclude that sequencing-based analysis may provide provisional grouping of SI-NETs by therapeutic targets or deregulated pathways. IntroductionSmall intestine neuroendocrine neoplasms (SI-NENs) are the most common malignancy of the small bowel, represent the largest group of NENs by organ site, and are studied in clinical treatment trials targeting PI3K/Akt/mTOR signaling. Whether this or other canonical cancer pathways is recurrently mutated, however, is uncertain, because a genome-wide, unbiased sequence analysis of cancer genes has not been performed to date in SI-NENs.Massively parallel, or "nextgen," DNA sequencing is currently advancing research in other human malignancies by facilitating the collection of comprehensive, genome-wide, unbiased datasets providing a common data framework for comparing results across different tumor types and gene sets. It provides the most comprehensive technology to date to explore the potential of genomics for individualizing cancer treatment within a tumor type. To unlock and explore the potential of the technology for translational research in SI-NEN, we sequenced 48 such tumors.