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We have shown previously the intercalation geometry of a series of acenaphtho [1,2-b] pyrrole derivatives with DNA double helix in vitro. In this report we chose a couple of intercalating analogues and a Chinese traditional medicine Tanshinone IIA as probes to investigate the response of DNA damage sensor ataxia-telangiectasia mutated (Atm) protein toward the DNA topological change in vivo. The two analogues (1)a (3-(4-Methyl-piperazin)-8-oxo-8H-acenaphtho [1,2-b]pyrrole -9-carbonitrile) and (3)a (3-(3-Dimethylamino-propylamino)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9- carbonitrile) could unwind double helix to different extents, whereas Tanshinone IIA could wind the double helix. Using a combination of circular dichroism (CD) studies and immunoflurescence assays, we found for the first time that the Atm protein kinase can respond to the unwinding chromatin conformational damage caused by (1)a and (3)a, while it could not be activated by the winding effects caused by Tanshinone IIA. Moreover, the amount of Atm protein phosphorylation is consistent with the degree of unwinding conformational damage. The average number of Atm foci in an MCF-7 cell is 32 +/- 1.5 at 6 microM (1)a, which is significantly higher than the 8 microM (3)a exposure (15 +/- 0.5, p < 0.5). A new couple of DNA topological probes, (1)a and (3)a have been found for the future semi-quantitative investigation of factors involved in the DNA damage pathway.
Mouse embryonic stem cells (mESC) are characterized by high proliferation activity. mESC are highly sensitive to genotoxic stresses and do not undergo G(1)/S checkpoint upon DNA-damage. mESC are supposed to develop sensitive mechanisms to maintain genomic integrity provided by either DNA damage repair or elimination of defected cells by apoptosis. The issue of how mESC recognize the damages and execute DNA repair remains to be studied. We analyzed the kinetics of DNA repair foci marked by antibodies to phosphorylated Atm kinase and histone H2AX (gammaH2AX). We showed that mESC display non-induced DNA single-strand breaks (SSBs), as revealed by comet-assay, and a noticeable background of gammaH2AX staining. Exposure of mESC to gamma-irradiation induced the accumulation of phosphorylated Atm-kinase in the nucleus as well as the formation of additional gammaH2AX foci, which disappeared thereafter. To decrease the background of gammaH2AX staining in control non-irradiated cells, we pre-synchronized mESC at the G(2)/M by low concentration of nocodazol for a short time (6 h). The cells were then irradiated and stained for gammaH2AX. Irradiation induced the formation of gammaH2AX foci both in G(2)-phase and mitotic cells, which evidenced for the active state of DNA-damage signaling at these stages of the cell cycle in mESC. Due to the G(1)/S checkpoint is compromised in mESCs, we checked, whether wild-type p53, a target for Atm kinase, was phosphorylated in response to gamma-irradiation. The p53 was barely phosphorylated in response to irradiation, which correlated with a very low expression of p53-target p21/Waf1 gene. Thus, in spite of the dysfunction of the p53/Waf1 pathway and the lack of cell cycle checkpoints, the mESC are capable of activating Atm and inducing gammaH2AX foci formation, which are necessary for the activation of DNA damage response.
This study identifies ataxia-telangiectasia mutated (Atm) as a further component of the complex signaling network of radiation-induced DNA damage in nontargeted bystander cells downstream of ataxia-telangiectasia and Rad3-related (ATR) and provides a rationale for molecular targeted modulation of these effects. In directly irradiated cells, ATR, Atm, and DNA-dependent protein kinase (DNA-PK) deficiency resulted in reduced cell survival as predicted by the known important role of these proteins in sensing DNA damage. A decrease in clonogenic survival was also observed in ATR/Atm/DNA-PK-proficient, nonirradiated bystander cells, but this effect was completely abrogated in ATR and Atm but not DNA-PK-deficient bystander cells. Atm activation in bystander cells was found to be dependent on ATR function. Furthermore, the induction and colocalization of ATR, 53BP1, Atm-S1981P, p21, and BRCA1 foci in nontargeted cells was shown, suggesting their involvement in bystander DNA damage signaling and providing additional potential targets for its modulation. 53BP1 bystander foci were induced in an ATR-dependent manner predominantly in S-phase cells, similar to gammaH2AX foci induction. In conclusion, these results provide a rationale for the differential modulation of targeted and nontargeted effects of radiation.
Cellular responses to DNA damage are crucial for maintaining genome integrity, virus infection, and preventing the development of cancer. Hepatitis C virus (HCV) infection and the expression of the HCV nonstructural protein NS3 and core protein have been proposed as factors involved in the induction of double-stranded DNA breaks and enhancement of the mutation frequency of cellular genes. Since DNA damage sensors, such as the ataxia-telangiectasia mutated kinase (Atm), Atm- and Rad3-related kinase (ATR), poly(ADP-ribose) polymerase 1 (PARP-1), and checkpoint kinase 2 (Chk2), play central roles in the response to genotoxic stress, we hypothesized that these sensors might affect HCV replication. To test this hypothesis, we examined the level of HCV RNA in HuH-7-derived cells stably expressing short hairpin RNA targeted to Atm, ATR, PARP-1, or Chk2. Consequently, we found that replication of both genome-length HCV RNA (HCV-O, genotype 1b) and the subgenomic replicon RNA were notably suppressed in Atm- or Chk2-knockdown cells. In addition, the RNA replication of HCV-JFH1 (genotype 2a) and the release of core protein into the culture supernatants were suppressed in these knockdown cells after inoculation of the cell culture-generated HCV. Consistent with these observations, Atm kinase inhibitor could suppress the HCV RNA replication. Furthermore, we observed that HCV NS3-NS4A interacted with Atm and that HCV NS5B interacted with both Atm and Chk2. Taken together, these results suggest that the Atm signaling pathway is critical for HCV RNA replication and may represent a novel target for the clinical treatment of patients with chronic hepatitis C.
Nonhomologous end joining (NHEJ) is the major pathway for the repair of DNA double strand breaks (DSBs) in human cells. NHEJ requires the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku70, Ku80, XRCC4, DNA ligase IV and Artemis, as well as DNA polymerases mu and lambda and polynucleotide kinase. Recent studies have identified an additional participant, XLF, for XRCC4-like factor (also called Cernunnos), which interacts with the XRCC4-DNA ligase IV complex and stimulates its activity in vitro, however, its precise role in the DNA damage response is not fully understood. Since the protein kinase activity of DNA-PKcs is required for NHEJ, we asked whether XLF might be a physiological target of DNA-PK. Here, we have identified two major in vitro DNA-PK phosphorylation sites in the C-terminal region of XLF, serines 245 and 251. We show that these represent the major phosphorylation sites in XLF in vivo and that serine 245 is phosphorylated in vivo by DNA-PK, while serine 251 is phosphorylated by Ataxia-Telangiectasia Mutated (Atm). However, phosphorylation of XLF did not have a significant effect on the ability of XLF to interact with DNA in vitro or its recruitment to laser-induced DSBs in vivo. Similarly, XLF in which the identified in vivo phosphorylation sites were mutated to alanine was able to complement the DSB repair defect as well as radiation sensitivity in XLF-deficient 2BN cells. We conclude that phosphorylation of XLF at these sites does not play a major role in the repair of IR-induced DSBs in vivo.
DNA damage induces hyper-phosphorylation of the Sp1 transcriptional factor. We have demonstrated that ionizing radiation-associated DNA double-strand breaks (DSBs) induce phosphorylation of at least Ser-56 and Ser-101 residues on Sp1 in an Atm-dependent manner. UV irradiation- or hydroxyurea (HU)-induced replicative stress results in phosphorylation of only the Ser-101 residue. Furthermore, silencing of the Atm- and Rad3-related protein (ATR) in Atm-deficient cells treated with HU abrogated the Ser-101 phosphorylation. Thus, phosphorylation of Ser-101 on Sp1 appears to be a general response to DNA damage dependent on both Atm and ATR. Although silencing of Sp1 expression by siRNA targeting resulted in an increase in sensitivity to ionizing radiation (IR), the Ser-101 phosphorylation did not affect transcriptional activity from the Sp1 responsive promoter. Confocal laser microscopy analysis revealed co-localization of phosphorylated Sp1 at Ser-101 with phosphorylated Atm at Ser-1981, the affected sites representing DSBs. These observations suggest that phosphorylated Sp1 might play a role in DNA repair at damage sites rather than functioning in transcriptional regulation.
In response to DNA damage, cells undergo either cell-cycle arrest or apoptosis, depending on the extent of damage and the cell's capacity for DNA repair. Cell-cycle arrest induced by double-stranded DNA breaks depends on activation of the ataxia-telangiectasia (Atm) protein kinase, which phosphorylates cell-cycle effectors such as Chk2 and p53 to inhibit cell-cycle progression. Atm is recruited to double-stranded DNA breaks by a complex of sensor proteins, including Mre11/Rad50/Nbs1, resulting in autophosphorylation, monomerization, and activation of Atm kinase.In characterizing Aven protein, a previously reported apoptotic inhibitor, we have found that Aven can function as an Atm activator to inhibit G2/M progression. Aven bound to Atm and Aven overexpressed in cycling Xenopus egg extracts prevented mitotic entry and induced phosphorylation of Atm and its substrates. Immunodepletion of endogenous Aven allowed mitotic entry even in the presence of damaged DNA, and RNAi-mediated knockdown of Aven in human cells prevented autophosphorylation of Atm at an activating site (S1981) in response to DNA damage. Interestingly, Aven is also a substrate of the Atm kinase. Mutation of Atm-mediated phosphorylation sites on Aven reduced its ability to activate Atm, suggesting that Aven activation of Atm after DNA damage is enhanced by Atm-mediated Aven phosphorylation.These results identify Aven as a new Atm activator and describe a positive feedback loop operating between Aven and Atm. In aggregate, these findings place Aven, a known apoptotic inhibitor, as a critical transducer of the DNA-damage signal.
A woman typically presents for genetic counselling because she has a strong family history and is interested in knowing the probability she will develop disease in the future; that is, her absolute risk. Relative risk for a given factor refers to risk compared with either population average risk (sense a), or risk when not having the factor, with all other factors held constant (sense b). Not understanding that these are three distinct concepts can result in failure to correctly appreciate the consequences of studies on clinical genetic testing. Several studies found that the frequencies of mutations in Atm, BRIP1, PALB2 and CHEK2 were many times greater for cases with a strong family history than for controls. To account for the selected case sampling (ascertainment), a statistical model that assumes that the effect of any measured variant multiplies the effect of unmeasured variants was applied. This multiplicative polygenic model in effect estimated the relative risk in the sense b, not sense a, and found it was in the range of 1.7 to 2.4. The authors concluded that the variants are "low penetrance". They failed to note that their model fits predicted that, for some women, absolute risk may be as high as for BRCA2 mutation carriers. This is because the relative risk multiplies polygenic risk, and the latter is predicted by family history. Therefore, mutation testing of these genes for women with a strong family history, especially if it is of early onset, may be as clinically relevant as it is for BRCA1 and BRCA2.
Human genomic instability syndromes affect the nervous system to different degrees of severity, attesting to the vulnerability of the CNS to perturbations of genomic integrity and the DNA damage response (DDR). Ataxia-telangiectasia (A-T) is a typical genomic instability syndrome whose major characteristic is progressive neuronal degeneration but is also associated with immunodeficiency, cancer predisposition and acute sensitivity to ionizing radiation and radiomimetic chemicals. A-T is caused by loss or inactivation of the Atm protein kinase, which mobilizes the complex, multi-branched cellular response to double strand breaks in the DNA by phosphorylating numerous DDR players. The link between Atm's function in the DDR and the neuronal demise in A-T has been questioned in the past. However, recent studies of the Atm-mediated DDR in neurons suggest that the neurological phenotype in A-T is indeed caused by deficiency in this function, similar to other features of the disease. Still, major issues concerning this phenotype remain open, including the presumed differences between the DDR in post-mitotic neurons and proliferating cells, the nature of the damage that accumulates in the DNA of Atm-deficient neurons under normal life conditions, the mode of death of Atm-deficient neurons, and the lack of a major neuronal phenotype in the mouse model of A-T. A-T remains a prototype disease for the study of the DDR's role in CNS development and maintenance.
Minutes after DNA damage, the variant histone H2AX is phosphorylated by protein kinases of the phosphoinositide kinase family, including Atm, ATR or DNA-PK. Phosphorylated (gamma)-H2AX-which recruits molecules that sense or signal the presence of DNA breaks, activating the response that leads to repair-is the earliest known marker of chromosomal DNA breakage. Here we identify a dynamic change in chromatin that promotes H2AX phosphorylation in mammalian cells. DNA breaks swiftly mobilize heterochromatin protein 1 (HP1)-beta (also called CBX1), a chromatin factor bound to histone H3 methylated on lysine 9 (H3K9me). Local changes in histone-tail modifications are not apparent. Instead, phosphorylation of HP1-beta on amino acid Thr 51 accompanies mobilization, releasing HP1-beta from chromatin by disrupting hydrogen bonds that fold its chromodomain around H3K9me. Inhibition of casein kinase 2 (CK2), an enzyme implicated in DNA damage sensing and repair, suppresses Thr 51 phosphorylation and HP1-beta mobilization in living cells. CK2 inhibition, or a constitutively chromatin-bound HP1-beta mutant, diminishes H2AX phosphorylation. Our findings reveal an unrecognized signalling cascade that helps to initiate the DNA damage response, altering chromatin by modifying a histone-code mediator protein, HP1, but not the code itself.