Yaniv Lerenthal

ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage

Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.

Requirement of the MRN complex for ATM activation by DNA damage

The ATM protein kinase is a primary activator of the cellular response to DNA double‐strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia‐telangiectasia (A‐T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A‐T like disease (A‐TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM‐mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A‐T and A‐TLD.

Mdc1 couples DNA double‐strand break recognition by Nbs1 with its H2AX‐dependent chromatin retention

Mdc1/NFBD1 controls cellular responses to DNA damage, in part via interacting with the Mre11–Rad50–Nbs1 complex that is involved in the recognition, signalling, and repair of DNA double‐strand breaks (DSBs). Here, we show that in live human cells, the transient interaction of Nbs1 with DSBs and its phosphorylation by ATM are Mdc1‐independent. However, ablation of Mdc1 by siRNA or mutation of the Nbs1s FHA domain required for Mdc1 binding reduced the affinity of Nbs1 for DSB‐flanking chromatin and caused aberrant pan‐nuclear dispersal of Nbs1. This occurred despite normal phosphorylation of H2AX, indicating that lack of Mdc1 does not impair this DSB‐induced chromatin change, but rather precludes the sustained engagement of Nbs1 with these regions. Mdc1 (but not Nbs1) became partially immobilized to chromatin after DSB generation, and siRNA‐mediated depletion of H2AX prevented such relocalization of Mdc1 and uncoupled Nbs1 from DSB‐flanking chromatin. Our data suggest that Mdc1 functions as an H2AX‐dependent interaction platform enabling a switch from transient, Mdc1‐independent recruitment of Nbs1 to DSBs towards sustained, Mdc1‐dependent interactions with the surrounding chromosomal microenvironment.

Requirement of ATM-Dependent Monoubiquitylation of Histone H2B for Timely Repair of DNA Double-Strand Breaks

The cellular response to DNA double-strand breaks (DSBs) is mobilized by the protein kinase ATM, which phosphorylates key players in the DNA damage response (DDR) network. A major question is how ATM controls DSB repair. Optimal repair requires chromatin relaxation at damaged sites. Chromatin reorganization is coupled to dynamic alterations in histone posttranslational modifications. Here, we show that in human cells, DSBs induce monoubiquitylation of histone H2B, a modification that is associated in undamaged cells with transcription elongation. We find that this process relies on recruitment to DSB sites and ATM-dependent phosphorylation of the responsible E3 ubiquitin ligase: the RNF20-RNF40 heterodimer. H2B monoubiquitylation is required for timely recruitment of players in the two major DSB repair pathways-nonhomologous end-joining and homologous recombination repair-and optimal repair via both pathways. Our data and previous data suggest a two-stage model for chromatin decondensation that facilitates DSB repair.

Ataxia Telangiectasia Mutated (ATM) Is Essential for DNA-PKcs Phosphorylations at the Thr-2609 Cluster upon DNA Double Strand Break

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is rapidly phosphorylated at the Thr-2609 cluster and Ser-2056 upon ionizing radiation (IR). Furthermore, DNA-PKcs phosphorylation at both regions is critical for its role in DNA double strand break (DSB) repair as well as cellular resistance to radiation. IR-induced DNA-PKcs phosphorylation at Thr-2609 and Ser-2056, however, exhibits distinct kinetics indicating that they are differentially regulated. Although DNA-PKcs autophosphorylates itself at Ser-2056 after IR, we have reported here that ATM mediates DNA-PKcs phosphorylation at Thr-2609 as well as at the adjacent (S/T)Q motifs within the Thr-2609 cluster. In addition, our data suggest that DNA-PKcs- and ATM-mediated DNA-PKcs phosphorylations are cooperative and required for the full activation of DNA-PKcs and the subsequent DSB repair. Elimination of DNA-PKcs phosphorylation at both regions severely compromises radioresistance and DSB repair. Finally, our result provides a possible mechanism for the direct involvement of ATM in non-homologous end joining-mediated DSB repair.

Involvement of Matrin 3 and SFPQ/NONO in the DNA damage response

The DNA damage response (DDR) is a complex signaling network that is induced by DNA lesions and vigorously activated by double strand breaks (DSBs). The DSB response is mobilized by the nuclear protein kinase ATM, which phosphorylates key players in its various branches. SFPQ (PSF) and NONO (p54) are nuclear proteins that interact with each other and have diverse roles in nucleic acids metabolism. The SFPQ/NONO heterodimer was previously found to enhance DNA strand break rejoining in vitro. Our attention was drawn to these two proteins as they interact with the nuclear matrix protein Matrin 3 (MATR3), which we found to be a novel ATM target. We asked whether SFPQ and NONO too are involved in the DSB response. Proteins that function at the early phase of this response are often recruited to the damaged sites. We observed rapid recruitment of SFPQ/NONO to sites of DNA damage induced by laser microbeam. In MATR3 knockdown cells SFPQ/NONO retention at DNA damage sites was prolonged. SFPQ and MATR3 depletion led to abnormal accumulation of cells at the S-phase of the cell cycle following treatment with the radiomimetic chemical neocarzinostatin. Notably, proteins involved in DSB repair via nonhomologous end-joining co-immunoprecipitated with NONO; SFPQ depletion delayed DSB repair. Collectively the data suggest that SFPQ, NONO and MATR3 are involved in the early stage of the DSB response, setting the scene for DSB repair.

Dissection of a DNA-damage-induced transcriptional network using a combination of microarrays, RNA interference and computational promoter analysis.

BackgroundGene-expression microarrays and RNA interferences (RNAi) are among the most prominent techniques in functional genomics. The combination of the two holds promise for systematic, large-scale dissection of transcriptional networks. Recent studies, however, raise the concern that nonspecific responses to small interfering RNAs (siRNAs) might obscure the consequences of silencing the gene of interest, throwing into question the ability of this experimental strategy to achieve precise network dissections.ResultsWe used microarrays and RNAi to dissect a transcriptional network induced by DNA damage in a human cellular system. We recorded expression profiles with and without exposure of the cells to a radiomimetic drug that induces DNA double-strand breaks (DSBs). Profiles were measured in control cells and in cells knocked-down for the Rel-A subunit of NFκB and for p53, two pivotal stress-induced transcription factors, and for the protein kinase ATM, the major transducer of the cellular responses to DSBs. We observed that NFκB and p53 mediated most of the damage-induced gene activation; that they controlled the activation of largely disjoint sets of genes; and that ATM was required for the activation of both pathways. Applying computational promoter analysis, we demonstrated that the dissection of the network into ATM/NFκB and ATM/p53-mediated arms was highly accurate.ConclusionsOur results demonstrate that the combined experimental strategy of expression arrays and RNAi is indeed a powerful method for the dissection of complex transcriptional networks, and that computational promoter analysis can provide a strong complementary means for assessing the accuracy of this dissection.

Transcriptional modulation induced by ionizing radiation: p53 remains a central player

The cellular response to DNA damage is vital for maintaining genomic stability and preventing undue cell death or cancer formation. The DNA damage response (DDR), most robustly mobilized by double‐strand breaks (DSBs), rapidly activates an extensive signaling network that affects numerous cellular systems, leading to cell survival or programmed cell death. A major component of the DDR is the widespread modulation of gene expression. We analyzed together six datasets that probed transcriptional responses to ionizing radiation (IR) – our novel experimental data and 5 published datasets – to elucidate the scope of this response and identify its gene targets. According to the mRNA expression profiles we recorded from 5 cancerous and non‐cancerous human cell lines after exposure to 5 Gy of IR, most of the responses were cell line‐specific. Computational analysis identified significant enrichment for p53 target genes and cell cycle‐related pathways among groups of up‐regulated and down‐regulated genes, respectively. Computational promoter analysis of the six datasets disclosed that a statistically significant number of the induced genes contained p53 binding site signatures. p53‐mediated regulation had previously been documented for subsets of these gene groups, making our lists a source of novel potential p53 targets. Real‐time qPCR and chromatin immunoprecipitation (ChIP) assays validated the IR‐induced p53‐dependent induction and p53 binding to the respective promoters of 11 selected genes. Our results demonstrate the power of a combined computational and experimental approach to identify new transcriptional targets in the DNA damage response network.

ATM-mediated phosphorylation of polynucleotide kinase/phosphatase is required for effective DNA double-strand break repair

The cellular response to double‐strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia‐telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5′‐OH and dephosphorylates 3′‐phosphate DNA ends that are formed at DSB termini caused by DNA‐damaging agents, thereby regenerating legitimate ends for further processing. We establish that the ATM phosphorylation targets on human PNKP—Ser 114 and Ser 126—are crucial for cellular survival following DSB induction and for effective DSB repair, being essential for damage‐induced enhancement of the activity of PNKP and its proper accumulation at the sites of DNA damage. These findings show a direct functional link between ATM and the DSB‐repair machinery.

ATM protein-dependent phosphorylation of Rad50 protein regulates DNA repair and cell cycle control

The Mre11/Rad50/NBN complex plays a central role in coordinating the cellular response to DNA double-strand breaks. The importance of Rad50 in that response is evident from the recent description of a patient with Rad50 deficiency characterized by chromosomal instability and defective ATM-dependent signaling. We report here that ATM (defective in ataxia-telangiectasia) phosphorylates Rad50 at a single site (Ser-635) that plays an important adaptor role in signaling for cell cycle control and DNA repair. Although a Rad50 phosphosite-specific mutant (S635G) supported normal activation of ATM in Rad50-deficient cells, it was defective in correcting DNA damage-induced signaling through the ATM-dependent substrate SMC1. This mutant also failed to correct radiosensitivity, DNA double-strand break repair, and an S-phase checkpoint defect in Rad50-deficient cells. This was not due to disruption of the Mre11/Rad50/NBN complex revealing for the first time that phosphorylation of Rad50 plays a key regulatory role as an adaptor for specific ATM-dependent downstream signaling through SMC1 for DNA repair and cell cycle checkpoint control in the maintenance of genome integrity.