Histone lysine methylation has been linked to the recruitment of mammalian DNA repair factor 53BP1 and putative fission yeast homolog Crb2 to DNA double-strand breaks (DSBs), but how histone recognition is achieved has not been established. Here we demonstrate that this link occurs through direct binding of 53BP1 and Crb2 to histone H4. Using X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, we show that, despite low amino acid sequence conservation, both 53BP1 and Crb2 contain tandem tudor domains that interact with histone H4 specifically dimethylated at Lys20 (H4-K20me2). The structure of 53BP1/H4-K20me2 complex uncovers a unique five-residue 53BP1 binding cage, remarkably conserved in the structure of Crb2, that best accommodates a dimethyllysine but excludes a trimethyllysine, thus explaining the methylation state-specific recognition of H4-K20. This study reveals an evolutionarily conserved molecular mechanism of targeting DNA repair proteins to DSBs by direct recognition of H4-K20me2.

H2AX, a member of the histone H2A family, is rapidly phosphorylated in response to ionizing radiation. This phosphorylation, at an evolutionary conserved C-terminal phosphatidylinositol 3-OH-kinase-related kinase (PI3KK) motif, is thought to be critical for recognition and repair of DNA double strand breaks. Here we report that inhibition of DNA replication by hydroxyurea or ultraviolet irradiation also induces phosphorylation and foci formation of H2AX. These phospho-H2AX foci colocalize with proliferating cell nuclear antigen (PCNA), BRCA1, and 53BP1 at the arrested replication fork in S phase cells. This response is ATR-dependent but does not require ATM or Hus1. Our findings suggest that, in addition to its role in the recognition and repair of double strand breaks, H2AX also participates in the surveillance of DNA replication.

Activation of the ataxia telangiectasia mutated (ATM) kinase triggers diverse cellular responses to ionizing radiation (IR), including the initiation of cell cycle checkpoints. Histone H2AX, p53 binding-protein 1 (53BP1) and Chk2 are targets of ATM-mediated phosphorylation, but little is known about their roles in signalling the presence of DNA damage. Here, we show that mice lacking either H2AX or 53BP1, but not Chk2, manifest a G2-M checkpoint defect close to that observed in ATM(-/-) cells after exposure to low, but not high, doses of IR. Moreover, H2AX regulates the ability of 53BP1 to efficiently accumulate into IR-induced foci. We propose that at threshold levels of DNA damage, H2AX-mediated concentration of 53BP1 at double-strand breaks is essential for the amplification of signals that might otherwise be insufficient to prevent entry of damaged cells into mitosis.

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.