Chk2-dependent phosphorylation of XRCC1 in the DNA damage response promotes base excision repair

The DNA damage response (DDR) has an essential function in maintaining genomic stability. Ataxia telangiectasia-mutated (ATM)-checkpoint kinase 2 (Chk2) and ATM- and Rad3-related (ATR)-Chk1, triggered, respectively, by DNA double-strand breaks and blocked replication forks, are two major DDRs processing structurally complicated DNA damage. In contrast, damage repaired by base excision repair (BER) is structurally simple, but whether, and how, the DDR is involved in repairing this damage is unclear. Here, we demonstrated that ATM-Chk2 was activated in the early response to oxidative and alkylation damage, known to be repaired by BER. Furthermore, Chk2 formed a complex with XRCC1, the BER scaffold protein, and phosphorylated XRCC1 in vivo and in vitro at Thr(284). A mutated XRCC1 lacking Thr(284) phosphorylation was linked to increased accumulation of unrepaired BER intermediate, reduced DNA repair capacity, and higher sensitivity to alkylation damage. In addition, a phosphorylation-mimic form of XRCC1 showed increased interaction with glycosylases, but not other BER proteins. Our results are consistent with the phosphorylation of XRCC1 by ATM-Chk2 facilitating recruitment of downstream BER proteins to the initial damage recognition/excision step to promote BER.     

Loss of RPA1 induces Chk2 phosphorylation through a caffeine-sensitive pathway

RPA is an important component of DNA replication, repair and recombination, but its involvement in the signaling of cell-cycle checkpoints is not well understood. In this study, we show that knockdown of RPA1 by siRNA duplexes induces ATM (Ser1981) and Chk2 (Thr68), but not Chk1 (Ser345) phosphorylation and results in p21 upregulation in HeLa cells. However, the induction of Chk2 (Thr68) phosphorylation and p21 expression by RPA1 siRNA transfection can be completely blocked by the ATM inhibitor caffeine. Moreover, transfection of siRNAs targeting ATM dramatically reduces Chk2 (Thr68) phosphorylation in RPA1 knockdown cells. Taken together, these results suggest that loss of RPA1 activates the Chk2 signaling pathway in an ATM-dependent manner.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

Distinct roles of ATR and DNA‐PKcs in triggering DNA damage responses in ATM‐deficient cells

The cellular response to DNA double‐strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell‐cycle‐dependent manner. Here, we report that the crucial checkpoint signalling proteins—p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2—are phosphorylated rapidly by ATR in an ATM/Mre11/cell‐cycle‐independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM‐deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM‐deficient cells enforce an early G2/M checkpoint that is ATR‐dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM‐deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP‐1), a protein involved in chromatin remodelling, is mediated by DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in a spatio‐temporal manner in addition to ATM. We posit that ATM substrates involved in cell‐cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA‐PKcs in addition to ATM.     

Molecular interaction map of the p53 and Mdm2 logic elements, which control the Off-On switch of p53 in response to DNA damage

The molecular network that controls responses to genotoxic stress is centered at p53 and Mdm2. Recent findings have shown this network to be more complex than previously envisioned. Using a notation specifically designed for circuit diagram-like representations of bioregulatory networks, we have prepared an updated molecular interaction map of the immediate connections of p53 and Mdm2, which are described as logic elements of the network. We use the map as the basis for a comprehensive review of current concepts of signal processing by these logic elements (an interactive version of the maps-eMIMs can be examined at ). We also used molecular interaction maps to propose a p53 Off-On switch in response to DNA damage.     

辐射诱导的染色质拓扑关联结构域层级变化及其在细胞辐射响应中的作用

基因组三维结构在基因表达调控中发挥重要作用,染色质拓扑关联结构域(topologically associated domain,TAD)是DNA复制和基因转录的基本功能单位,也是DNA损伤修复的功能单元,在辐射诱导的DNA损伤修复中发挥重要作用。近期研究表明,TAD并非是完全独立的结构单元,其内部常呈现多层级结构,对基因表达具有重要调控作用。为探究TAD多层级结构在细胞辐射响应中的作用,本研究使用TAD层级结构识别算法OnTAD对Gene expression omnibus数据库中5Gy X射线照射的淋巴细胞、成纤维细胞和毛细血管扩张性共济失调突变(ataxia telangiectasia mutated,ATM)基因缺陷的成纤维细胞,共26个样本的Hi-C(high-through chromosome conformation capture,Hi-C)数据进行分析,发现辐射后细胞的TAD层级结构出现规律性变化,高层级TAD缺失较多,低层级TAD相对保守;辐射诱导的TAD层级结构变化通过调节基因表达参与细胞辐射响应;ATM是辐射诱导TAD层级结构变化和恢复的重要因子。本研究为从TAD多层级结构角度理解基因组三维结构在细胞辐射响应中的作用提供了新思路。     

Taking the time to make important decisions: The checkpoint effector kinases Chk1 and Chk2 and the DNA damage response

The cellular DNA damage response (DDR) is activated by many types of DNA lesions. Upon recognition of DNA damage by sensor proteins, an intricate signal transduction network is activated to coordinate diverse cellular outcomes that promote genome integrity. Key components of the DDR in mammalian cells are the checkpoint effector kinases Chk1 and Chk2 (referred to henceforth as the effector kinases; orthologous to spChk1 and spCds1 in the fission yeast S. pombe and scChk1 and scRad53 in the budding yeast S. cerevisiae). These evolutionarily conserved and structurally divergent kinases phosphorylate numerous substrates to regulate the DDR. This review will focus on recent advances in our understanding of the structure, regulation, and functions of the effector kinases in the DDR, as well as their potential roles in human disease.     

Phosphorylation of SMURF2 by ATM exerts a negative feedback control of DNA damage response

Timely repair of DNA double-strand breaks (DSBs) is essential to maintaining genomic integrity and preventing illnesses induced by genetic abnormalities. We previously demonstrated that the E3 ubiquitin ligase SMURF2 plays a critical tumor suppressing role via its interaction with RNF20 (ring finger protein 20) in shaping chromatin landscape and preserving genomic stability. However, the mechanism that mobilizes SMURF2 in response to DNA damage remains unclear. Using biochemical approaches and MS analysis, we show that upon the onset of the DNA-damage response, SMURF2 becomes phosphorylated at Ser³⁸⁴ by ataxia telangiectasia mutated (ATM) serine/threonine kinase, and this phosphorylation is required for its interaction with RNF20. We demonstrate that a SMURF2 mutant with an S384A substitution has reduced capacity to ubiquitinate RNF20 while promoting Smad3 ubiquitination unabatedly. More importantly, mouse embryonic fibroblasts expressing the SMURF2 S384A mutant show a weakened ability to sustain the DSB response compared with those expressing WT SMURF2 following etoposide treatment. These data indicate that SMURF2-mediated RNF20 ubiquitination and degradation controlled by ataxia telangiectasia mutated–induced phosphorylation at Ser³⁸⁴ constitutes a negative feedback loop that regulates DSB repair.     

Structure of the human dimeric ATM kinase

DNA-double strand breaks activate the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) to initiate DNA damage signal transduction. This activation process involves autophosphorylation and dissociation of inert ATM dimers into monomers that are catalytically active. Using single-particle electron microscopy (EM), we determined the structure of dimeric ATM in its resting state. The EM map could accommodate the crystal structure of the N-terminal truncated mammalian target of rapamycin (mTOR), a closely related enzyme of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, allowing for the localization of the N- and the C-terminal regions of ATM. In the dimeric structure, the actives sites are buried, restricting the access of the substrates to these sites. The unanticipated domain organization of ATM provides a basis for understanding its mechanism of inhibition.     

The protein phosphatase 1 regulator PNUTS is a new component of the DNA damage response

The function of protein phosphatase 1 nuclear-targeting subunit (PNUTS)--one of the most abundant nuclear-targeting subunits of protein phosphatase 1 (PP1c)--remains largely uncharacterized. We show that PNUTS depletion by small interfering RNA activates a G2 checkpoint in unperturbed cells and prolongs G2 checkpoint and Chk1 activation after ionizing-radiation-induced DNA damage. Overexpression of PNUTS-enhanced green fluorescent protein (EGFP)--which is rapidly and transiently recruited at DNA damage sites--inhibits G2 arrest. Finally, γH2AX, p53-binding protein 1, replication protein A and Rad51 foci are present for a prolonged period and clonogenic survival is decreased in PNUTS-depleted cells after ionizing radiation treatment. We identify the PP1c regulatory subunit PNUTS as a new and integral component of the DNA damage response involved in DNA repair.     

Phosphoproteomic analysis reveals plant DNA damage signalling pathways with a functional role for histone H2AX phosphorylation in plant growth under genotoxic stress

DNA damage responses are crucial for plant growth under genotoxic stress. Accumulating evidence indicates that DNA damage responses differ between plant cell types. Here, quantitative shotgun phosphoproteomics provided high‐throughput analysis of the DNA damage response network in callus cells. MS analysis revealed a wide network of highly dynamic changes in the phosphoprotein profile of genotoxin‐treated cells, largely mediated by the ATAXIA TELANGIECTASIA MUTATED (ATM) protein kinase, representing candidate factors that modulate plant growth, development and DNA repair. A C‐terminal dual serine target motif unique to H2AX in the plant lineage showed 171‐fold phosphorylation that was absent in atm mutant lines. The physiological significance of post‐translational DNA damage signalling to plant growth and survival was demonstrated using reverse genetics and complementation studies of h2ax mutants, establishing the functional role of ATM‐mediated histone modification in plant growth under genotoxic stress. Our findings demonstrate the complexity and functional significance of post‐translational DNA damage signalling responses in plants and establish the requirement of H2AX phosphorylation for plant survival under genotoxic stress.     

Synthesis and preliminary structure-activity relationship study of 2-aryl-2H-pyrazolo[4,3-c]quinolin-3-ones as potential checkpoint kinase 1 (Chk1) inhibitors

The serine-threonine checkpoint kinase 1 (Chk1) plays a critical role in the cell cycle arrest in response to DNA damage. In the last decade, Chk1 inhibitors have emerged as a novel therapeutic strategy to potentiate the anti-tumour efficacy of cytotoxic chemotherapeutic agents. In the search for new Chk1 inhibitors, a congeneric series of 2-aryl-2?H-pyrazolo[4,3-c]quinolin-3-one (PQ) was evaluated by in-vitro and in-silico approaches for the first time. A total of 30 PQ structures were synthesised in good to excellent yields using conventional or microwave heating, highlighting that 14 of them are new chemical entities. Noteworthy, in this preliminary study two compounds 4e₂ and 4h₂ have shown a modest but significant reduction in the basal activity of the Chk1 kinase. Starting from these preliminary results, we have designed the second generation of analogous in this class and further studies are in progress in our laboratories.     

Genome-wide screen of human bromodomain-containing proteins identifies Cecr2 as a novel DNA damage response protein

The formation of γ-H2AX foci after DNA double strand breaks (DSBs) is crucial for the cellular response to this lethal DNA damage. We previously have shown that BRG1, a chromatin remodeling enzyme, facilitates DSB repair by stimulating γ-H2AX formation, and this function of BRG1 requires the binding of BRGI to acetylated histone H3 on γ-H2AX-containing nucleosomes using its bromodomain (BRD), a protein module that specifically recognizes acetyl-Lys moieties. We also have shown that the BRD of BRG1, when ectopically expressed in cells, functions as a dominant negative inhibitor of the BRG1 activity to stimulate γ-H2AX and DSB repair. Here, we found that BRDs from a select group of proteins have no such activity, suggesting that the γ-H2AX inhibition activity of BRG1 BRD is specific. This finding led us to search for more BRDs that exhibit γ-H2AX inhibition activity in the hope of finding additional BRD-containing proteins involved in DNA damage responses. We screened a total of 52 individual BRDs present in 38 human BRD-containing proteins, comprising 93% of all human BRDs. We identified the BRD of cat eye syndrome chromosome region candidate 2 (Cecr2), which recently was shown to form a novel chromatin remodeling complex with unknown cellular functions, as having a strong γ-H2AX inhibition activity. This activity of Cecr2 BRD is specific because it depends on the chromatin binding affinity of Cecr2 BRD. Small interfering RNA knockdown experiments showed that Cecr2 is important for γ-H2AX formation and DSB repair. Therefore, our genomewide screen identifies Cecr2 as a novel DNA damage response protein.     

Modulation of DNA damage response and induction of apoptosis mediates synergism between doxorubicin and a new imidazopyridine derivative in breast and lung cancer cells

DNA damage response machinery (DDR) is an attractive target of cancer therapy. Modulation of DDR network may alter the response of cancer cells to DNA damaging anticancer drugs such as doxorubicin. The aim of the present study is to investigate the effects of a newly developed imidazopyridine (IAZP) derivative on the DDR after induction of DNA damage in cancer cells by doxorubicin. Cytotoxicity sulphrhodamine-B assay showed a weak anti-proliferative effect of IAZP alone on six cancer cell lines (MCF7, A549, A549DOX11, HepG2, HeLa and M8) and a normal fibroblast strain. Combination of IAZP with doxorubicin resulted in synergism in lung (A549) and breast (MCF7) cancer cells but neither in the other cancer cell lines nor in normal fibroblasts. Molecular studies revealed that synergism is mediated by modulation of DNA damage response and induction of apoptosis. Using constant-field gel electrophoresis and immunofluorescence detection of γ-H2AX foci, IAZP was shown to inhibit the repair of doxorubicin-induced DNA damage in A549 and MCF7 cells. Immunoblot analysis showed that IAZP suppresses the phosphorylation of the ataxia lelangiectasia and Rad3 related (ATR) protein, which is an important player in the response of cancer cells to chemotherapy-induced DNA damage. Moreover, IAZP augmented the doxorubicin-induced degradation of p21, activation of p53, CDK2, caspase 3/7 and phosphorylation of Rb protein. These effects enhanced doxorubicin-induced apoptosis in both cell lines. Our results indicate that IAZP is a promising agent that may enhance the cytotoxic effects of doxorubicin on some cancer cells through targeting the DDR. It is a preliminary step toward the clinical application of IAZP in combination with anticancer drugs and opens the avenue for the development of compounds targeting the DDR pathway that might improve the therapeutic index of anticancer drugs and enhance their cure rate.