Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX

In mammalian cells, DNA double-strand breaks (DSBs) cause rapid phosphorylation of the H2AX core histone variant (to form gamma-H2AX) in megabase chromatin domains flanking sites of DNA damage. To investigate the role of H2AX in mammalian cells, we generated H2AX-deficient (H2AX(Delta)/Delta) mouse embryonic stem (ES) cells. H2AX(Delta)/Delta ES cells are viable. However, they are highly sensitive to ionizing radiation (IR) and exhibit elevated levels of spontaneous and IR-induced genomic instability. Notably, H2AX is not required for NHEJ per se because H2AX(Delta)/Delta ES cells support normal levels and fidelity of V(D)J recombination in transient assays and also support lymphocyte development in vivo. However, H2AX(Delta)/Delta ES cells exhibit altered IR-induced BRCA1 focus formation. Our findings indicate that H2AX function is essential for mammalian DNA repair and genomic stability.     

Bioinformatic identification of genes suppressing genome instability

Unbiased forward genetic screens for mutations causing increased gross chromosomal rearrangement (GCR) rates in Saccharomyces cerevisiae are hampered by the difficulty in reliably using qualitative GCR assays to detect mutants with small but significantly increased GCR rates. We therefore developed a bioinformatic procedure using genome-wide functional genomics screens to identify and prioritize candidate GCR-suppressing genes on the basis of the shared drug sensitivity suppression and similar genetic interactions as known GCR suppressors. The number of known suppressors was increased from 75 to 110 by testing 87 predicted genes, which identified unanticipated pathways in this process. This analysis explicitly dealt with the lack of concordance among high-throughput datasets to increase the reliability of phenotypic predictions. Additionally, shared phenotypes in one assay were imperfect predictors for shared phenotypes in other assays, indicating that although genome-wide datasets can be useful in aggregate, caution and validation methods are required when deciphering biological functions via surrogate measures, including growth-based genetic interactions.     

Evidence of DNA damage in Alzheimer disease: phosphorylation of histone H2AX in astrocytes

Phosphorylation of the histone family is not only a response to cell signaling stimuli, but also an important indicator of DNA damage preceding apoptotic changes. While astrocytic degeneration, including DNA damage, has been reported in Alzheimer disease (AD), its pathogenetic significance is somewhat unclear. In an effort to clarify this, we investigated the expression of γH2AX as evidence of DNA damage in astrocytes to elucidate the role of these cells in the pathogenesis of AD. In response to the formation of double-stranded breaks in chromosomal DNA, serine 139 on H2AX, a 14-kDa protein that is a member of the H2A histone family and part of the nucleosome structure, becomes rapidly phosphorylated to generate γH2AX. Using immunocytochemical techniques, we found significantly increased levels of γH2AX in astrocytes in regions know to be vulnerable in AD, i.e., the hippocampal regions and cerebral cortex. These results suggest that astrocytes contain DNA damage, possibly resulting in functional disability, which in turn reduces their support for neurons. These findings further define the role of astrocyte dysfunction in the progression of AD.     

Cell cycle restriction by histone H2AX limits proliferation of adult neural stem cells

Adult neural stem cell proliferation is dynamic and has the potential for massive self-renewal yet undergoes limited cell division in vivo. Here, we report an epigenetic mechanism regulating proliferation and self-renewal. The recruitment of the PI3K-related kinase signaling pathway and histone H2AX phosphorylation following GABA(A) receptor activation limits subventricular zone proliferation. As a result, NSC self-renewal and niche size is dynamic and can be directly modulated in both directions pharmacologically or by genetically targeting H2AX activation. Surprisingly, changes in proliferation have long-lasting consequences on stem cell numbers, niche size, and neuronal output. These results establish a mechanism that continuously limits proliferation and demonstrates its impact on adult neurogenesis. Such homeostatic suppression of NSC proliferation may contribute to the limited self-repair capacity of the damaged brain.     

Germ-line variant of human NTH1 DNA glycosylase induces genomic instability and cellular transformation

Base excision repair (BER) removes at least 20,000 DNA lesions per human cell per day and is critical for the maintenance of genomic stability. We hypothesize that aberrant BER, resulting from mutations in BER genes, can lead to genomic instability and cancer. The first step in BER is catalyzed by DNA N-glycosylases. One of these, n^th endonuclease III-like (NTH1), removes oxidized pyrimidines from DNA, including thymine glycol. The rs3087468 single nucleotide polymorphism of the NTH1 gene is a G-to-T base substitution that results in the NTH1 D239Y variant protein that occurs in ∼6.2% of the global population and is found in Europeans, Asians, and sub-Saharan Africans. In this study, we functionally characterize the effect of the D239Y variant expressed in immortal but nontransformed human and mouse mammary epithelial cells. We demonstrate that expression of the D239Y variant in cells also expressing wild-type NTH1 leads to genomic instability and cellular transformation as assessed by anchorage-independent growth, focus formation, invasion, and chromosomal aberrations. We also show that cells expressing the D239Y variant are sensitive to ionizing radiation and hydrogen peroxide and accumulate double strand breaks after treatment with these agents. The DNA damage response is also activated in D239Y-expressing cells. In combination, our data suggest that individuals possessing the D239Y variant are at risk for genomic instability and cancer.     

H_2O_2致小鼠淋巴细胞DNA的损伤及修复

目的　应用单细胞电泳检测 (SCGE)过氧化氢 (H2 O2 )致小鼠淋巴细胞 DNA损伤及其修复。方法　标准 SCGE条件下荧光显微镜观察。结果　 H2 O2 不同浓度 80、16 0和 32 0μmol/ L ,3个组 H2 O2 浓度均可致小鼠淋巴细胞 DNA损伤 ,较低剂量即可造成细胞拖尾 ,可逆性损伤在 6 0 m in内可获得明显修复。结论　单细胞电泳可快速灵敏地检测氧化损伤 ,也可能用于流行病学研究。     

Determinants of spontaneous mutation in the bacterium Escherichia coli as revealed by whole-genome sequencing

A complete understanding of evolutionary processes requires that factors determining spontaneous mutation rates and spectra be identified and characterized. Using mutation accumulation followed by whole-genome sequencing, we found that the mutation rates of three widely diverged commensal Escherichia coli strains differ only by about 50%, suggesting that a rate of 1–2 × 10⁻³ mutations per generation per genome is common for this bacterium. Four major forces are postulated to contribute to spontaneous mutations: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage caused by exogenous agents, and the activities of error-prone polymerases. To determine the relative importance of these factors, we studied 11 strains, each defective for a major DNA repair pathway. The striking result was that only loss of the ability to prevent or repair oxidative DNA damage significantly impacted mutation rates or spectra. These results suggest that, with the exception of oxidative damage, endogenously induced DNA damage does not perturb the overall accuracy of DNA replication in normally growing cells and that repair pathways may exist primarily to defend against exogenously induced DNA damage. The thousands of mutations caused by oxidative damage recovered across the entire genome revealed strong local-sequence biases of these mutations. Specifically, we found that the identity of the 3′ base can affect the mutability of a purine by oxidative damage by as much as eightfold.A complete understanding of the evolution and stability of the genome requires that the determinants of spontaneous mutation be identified and characterized. Among the variety of mistakes that can occur during DNA transactions, four sources of sequence variation appear to dominate in prokaryotes: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage induced by exogenous agents, and the activities of error-prone polymerases. This conclusion is based on changes in the rates and spectra of mutations that occur when genes affecting these processes are deleted or amplified. In particular, loss of a DNA repair pathway often gives a mutator phenotype, indicating that the pathway of interest exerts an important limitation on spontaneous mutation (1). However, investigations of the mutagenic impact of various DNA repair pathways have relied almost exclusively on reporter genes, leaving open the possibility that the results are biased by the particular features of the selected loci. This concern can be avoided by allowing mutations to accumulate nonselectively in DNA repair-defective strains and identifying the resulting sequence changes by whole-genome sequencing (WGS). Although this approach may miss rare but interesting mutational processes, it can reveal the overall threats to genomic stability and identify features, such as local sequence context, that influence mutational frequencies. Surprisingly, this technique has been used with the eukaryote Caenorhabditis elegans (2) but has not been extensively applied to prokaryotes.The mutation accumulation (MA) protocol involves establishing multiple clonal populations from a single founder and then repeatedly passing the lines through single-individual bottlenecks (3, 4), which in bacteria is achieved easily by streaking for single colonies on agar medium. This procedure allows mutations to accumulate in an unbiased manner with a minimum of selective pressure. After a sufficient number of generations have occurred, the genomes are sequenced, and mutations are identified. Using this technique, we recently determined the intrinsic mutation rates and mutational spectra of repair-proficient strains of Escherichia coli and Bacillus subtilis and documented the mutational impact of the loss of the major error-correcting system, mismatch repair (MMR) (5–7). In the studies reported here we concentrate on E. coli, first asking if other commensal strains of E. coli have the same mutation rate and spectrum as our K12 strain and whether changing the growth medium influences mutation. Then we determined the mutational effects of the loss of several important DNA repair pathways. Our major conclusion is that, under the conditions of our experiments, mutation rates and spectra are nearly impervious to the loss of DNA repair functions except for those that deal with oxidative DNA damage. We also show that the mutagenicity of a major oxidative lesion, 7,8-dihydro-8-oxoguanine (8-oxoG), is highly dependent on the local sequence context.     

Role of tyrosyl-DNA phosphodiesterase (TDP1) in mitochondria

Human tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond at a DNA 3'-end linked to a tyrosyl moiety and has been implicated in the repair of topoisomerase I (Top1)-DNA covalent complexes. TDP1 can also hydrolyze other 3'-end DNA alterations including 3'-phosphoglycolate and 3'-abasic sites, and exhibits 3'-nucleosidase activity indicating it may function as a general 3'-end-processing DNA repair enzyme. Here, using laser confocal microscopy, subcellular fractionation and biochemical analyses we demonstrate that a fraction of the TDP1 encoded by the nuclear TDP1 gene localizes to mitochondria. We also show that mitochondrial base excision repair depends on TDP1 activity and provide evidence that TDP1 is required for efficient repair of oxidative damage in mitochondrial DNA. Together, our findings provide evidence for TDP1 as a novel mitochondrial enzyme.     

Loss of autophagy causes a synthetic lethal deficiency in DNA repair

(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the error-free DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.The preservation of genome integrity is critical for the prevention of human disease. In addition, the maintenance of proteome integrity is also considered central to healthy cellular homeostasis. Macroautophagy, hereafter referred to as autophagy, is a process that is paramount in counteracting damage to cytoplasmic constituents (1). Upon initiation of autophagy, double-membraned vesicles termed “autophagosomes” form to encapsulate cargoes including damaged or misfolded proteins and organelles. These vesicles ultimately fuse with lysosomes and the acidic hydrolases provided by the lysosome degrade cargoes into constituent parts, which can be recycled into biosynthetic pathways or in some situations, further catabolized to produce energy for the cell (1). Autophagy functions at basal levels in virtually all cells and is a major mechanism for protein turnover and the only known mechanism for degradation of organelles (1). Due to its crucial role in maintaining cytoplasmic and therefore cellular homeostasis, perturbations in autophagy have been reported to be an important contributing factor in a spectrum of diseases, including Crohn’s disease, lysosomal storage disorders, neurodegenerative diseases, and cancer (2–6).Autophagy operates in the cytoplasm and yet studies have shown that autophagy-deficient cells accumulate DNA damage (5). The reasons behind this observation, however, are not completely clear. Because the cellular environment of autophagy-deficient cells will cause accrual of damaged proteins with abnormal function and as a result accumulation of reactive oxygen species, it is easily conceivable that this will ultimately lead to a higher incidence of genetic lesions. However, even when autophagy is competent, our cells are already subject to an extremely high frequency of spontaneous DNA damage. The fact that this damage does not persist is due to highly efficient processes of DNA repair that serve to maintain genomic integrity (7, 8). We hypothesized, therefore, that the accumulation of genetic lesions in autophagy-deficient cells may be critically driven by a defect in DNA repair. We show that loss of autophagy leads to decreased levels of checkpoint kinase 1 (Chk1) and a greatly diminished ability to repair DNA double-strand breaks by homologous recombination (HR). As a result, autophagy-deficient cells are more reliant on nonhomologous end joining (NHEJ) for DNA repair, which uncovers a unique synthetic lethality-based strategy to kill cells that may be applicable to the treatment of various forms of human disease.     

ATM signals to TSC2 in the cytoplasm to regulate mTORC1 in response to ROS

Ataxia-telangiectasia mutated (ATM) is a cellular damage sensor that coordinates the cell cycle with damage-response checkpoints and DNA repair to preserve genomic integrity. However, ATM also has been implicated in metabolic regulation, and ATM deficiency is associated with elevated reactive oxygen species (ROS). ROS has a central role in many physiological and pathophysiological processes including inflammation and chronic diseases such as atherosclerosis and cancer, underscoring the importance of cellular pathways involved in redox homeostasis. We have identified a cytoplasmic function for ATM that participates in the cellular damage response to ROS. We show that in response to elevated ROS, ATM activates the TSC2 tumor suppressor via the LKB1/AMPK metabolic pathway in the cytoplasm to repress mTORC1 and induce autophagy. Importantly, elevated ROS and dysregulation of mTORC1 in ATM-deficient cells is inhibited by rapamycin, which also rescues lymphomagenesis in Atm-deficient mice. Our results identify a cytoplasmic pathway for ROS-induced ATM activation of TSC2 to regulate mTORC1 signaling and autophagy, identifying an integration node for the cellular damage response with key pathways involved in metabolism, protein synthesis, and cell survival.     

韩宗超

目的 观测过氧化氢诱导的大鼠衰老神经细胞DNA损伤与修复情况。方法 在荧光显微镜下观察不同浓度H2O2处理的3月、8月和26月龄大鼠的脑皮质、海马和基底节经分离的神经细胞及氧化损伤断裂的单链DNA,并做彗星图像分析。结果 在相同浓度H2O2介导下,衰老神经细胞DNA损伤分值较正常神经细胞高,皮质细胞较海马、基底节区细胞DNA损伤分值高,细胞更易受损伤。给予0．5～4．0 h孵育后,表明脑细胞最大修复时间仅为1．0 h,且衰老神经细胞较正常神经细胞的DNA损伤分值高,修复能力下降。当H2O2介导浓度为64 μmol／L时,3月龄鼠脑皮质、海马和基底节神经细胞DNA损伤分值依次为250、213和205,26月龄鼠DNA损伤分值依次为355、340和335。当H2O2介导浓度为128 μmol／L时,26月龄鼠DNA修复率约为10％。结论 彗星实验是一种非常敏感的检测DNA损伤与修复的方法。     

Modulation of mismatch repair and genomic stability by miR-155

Inactivation of mismatch repair (MMR) is the cause of the common cancer predisposition disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC), as well as 10–40% of sporadic colorectal, endometrial, ovarian, gastric, and urothelial cancers. Elevated mutation rates (mutator phenotype), including simple repeat instability [microsatellite instability (MSI)] are a signature of MMR defects. MicroRNAs (miRs) have been implicated in the control of critical cellular pathways involved in development and cancer. Here we show that overexpression of miR-155 significantly down-regulates the core MMR proteins, hMSH2, hMSH6, and hMLH1, inducing a mutator phenotype and MSI. An inverse correlation between the expression of miR-155 and the expression of MLH1 or MSH2 proteins was found in human colorectal cancer. Finally, a number of MSI tumors with unknown cause of MMR inactivation displayed miR-155 overexpression. These data provide support for miR-155 modulation of MMR as a mechanism of cancer pathogenesis.     

Nucleostemin deletion reveals an essential mechanism that maintains the genomic stability of stem and progenitor cells

Stem and progenitor cells maintain a robust DNA replication program during the tissue expansion phase of embryogenesis. The unique mechanism that protects them from the increased risk of replication-induced DNA damage, and hence permits self-renewal, remains unclear. To determine whether the genome integrity of stem/progenitor cells is safeguarded by mechanisms involving molecules beyond the core DNA repair machinery, we created a nucleostemin (a stem and cancer cell-enriched protein) conditional-null allele and showed that neural-specific knockout of nucleostemin predisposes embryos to spontaneous DNA damage that leads to severe brain defects in vivo. In cultured neural stem cells, depletion of nucleostemin triggers replication-dependent DNA damage and perturbs self-renewal, whereas overexpression of nucleostemin shows a protective effect against hydroxyurea-induced DNA damage. Mechanistic studies performed in mouse embryonic fibroblast cells showed that loss of nucleostemin triggers DNA damage and growth arrest independently of the p53 status or rRNA synthesis. Instead, nucleostemin is directly recruited to DNA damage sites and regulates the recruitment of the core repair protein, RAD51, to hydroxyurea-induced foci. This work establishes the primary function of nucleostemin in maintaining the genomic stability of actively dividing stem/progenitor cells by promoting the recruitment of RAD51 to stalled replication-induced DNA damage foci.     

缺锌对衰老小鼠抗氧化系统和肝脏DNA损伤修复功能的影响

目的　通过D 半乳糖诱导小鼠衰老模型 ,探讨缺锌对衰老小鼠抗氧化系统和肝脏DNA损伤与修复的影响。　方法　雄性 3月龄小鼠 70只 ,随机分成 5组 :青年组、衰老模型组、衰老缺锌组、衰老配喂组和衰老补锌组。各衰老组按 10 0mg /kg经颈背部皮下给予D 半乳糖注射液 ,青年组给予等剂量的生理盐水 ,连续 30d。衰老缺锌组和补锌组喂饲缺锌饲料 (含锌 1 6 1μg/kg) ,其他组喂饲正常锌饲料 (含锌 5 0 μg/kg) ,最后 2周补锌组喂饲补锌饲料 (10 0 μg/kg)。第 30天处死小鼠 ,取样检测血清锌、肝锌、超氧化物歧化酶、丙二醇、肝脂褐质和DNA损伤情况。　结果　与衰老模型组相比 ,衰老缺锌组血清锌 (0 5 3± 0 1)mg/L、肝锌 (14 5 4± 2 18)mg/L水平下降 ,血清和肝超氧化物歧化酶活性〔(14 2 87± 10 16 )NU/ml和 (180 11± 13 2 2 )NU/ml,P <0 0 5〕降低 ,丙二醇含量升高 ,肝脂褐质含量增高 ;彗星试验显示衰老缺锌组小鼠肝DNA损伤加重 ,彗星细胞尾长 /总长比值显著增加。补锌后上述指标均有改善。　结论　锌可有效的影响衰老的速度和程度 ,缺锌可加速衰老的进程 ,适当补锌有助于延缓衰老。     

Comparative levels of DNA breaks and sensitivity to oxidative stress in aged and senescent human fibroblasts: a distinctive pattern for centenarians

Basal and H₂O₂-induced DNA breaks as well as DNA repair activity and efficacy of the antioxygenic system were determined in human dermal fibroblasts explanted from either (i) young donors and passaged serially to reach replicative senescence or (ii) young, old and centenarian donors and shortly propagated in culture. These fibroblasts have been employed as an in vitro and ex vivo model, respectively, to evaluate comparatively DNA integrity during senescence (increasing population doubling levels) and aging (increasing donor age). Constitutive levels of DNA total strand breaks, as determined by the alkaline extraction procedure, changed moderately among the different cell lines, which exhibited, however, significant differences in the amount of either single or double strand breaks. The former decreased along with both aging and senescence; the latter augmented during senescence while being virtually steady during aging. Moreover, fibroblasts from centenarians showed to be less sensitive to H₂O₂-induced DNA damage than otherex vivo fibroblasts. This feature could not account for either increased DNA repair activity or higher efficacy of the antioxygenic system and pointed, instead, to an intrinsic nuclear stability which might be typical of centenarian fibroblasts and potentially functional to longevity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Oxidative DNA damage in human esophageal cancer: clinicopathological analysis of 8‐hydroxydeoxyguanosine and its repair enzyme

Both internal and external oxidative stresses act on DNA and can induce carcinogenesis. 8‐hydroxydeoxyguanosine (8‐OHdG) is an indicator of oxidative stress and it leads to transversion mutations and carcinogenesis. 8‐OHdG is excision‐repaired by 8‐OHdG DNA glycosylase (OGG1). The purpose of this study is to clarify the effect of oxidative DNA damage and repair enzymes on esophageal carcinogenesis. The levels of 8‐OHdG and OGG1 were immunohistochemically evaluated in resected specimens, including squamous cell carcinoma (SCC) in 97 patients with esophageal cancer. Higher levels of 8‐OHdG in normal esophageal epithelium were associated with a higher smoking index (P = 0.0464). The 8‐OHdG level was higher in cancerous areas than in normal epithelia (P = 0.0061), whereas OGG1 expression was weaker in cancerous areas than in normal epithelia (P < 0.0001). An increase of OGG1 expression in normal epithelium was observed as 8‐OHdG levels increased (P = 0.0011). However, this correlation was not observed in cancerous areas. High OGG1 expression in the cytoplasm was related to deeper tumors (P = 0.0023), node metastasis (P = 0.0065) and stage (P = 0.0019). Oxidative DNA damage, which is attributable to smoking as well as disturbances in DNA repair systems, appears to be closely related to esophageal carcinogenesis and its progression.     

Measurement of 7,8-dihydro-8-oxo-2'-deoxyguanosine metabolism in MCF-7 cells at low concentrations using accelerator mass spectrometry

Growing evidence suggests that oxidative damage to cells generates mutagenic 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), which may initiate diseases related to aging and carcinogenesis. Kinetic measurement of 8-oxodG metabolism and repair in cells has been hampered by poor assay sensitivity and by difficulty characterizing the flux of oxidized nucleotides through the relevant metabolic pathways. We report here the development of a sensitive and quantitative approach to characterizing the kinetics and metabolic sources of 8-oxodG in MCF-7 human breast cancer cells by accelerator mass spectrometry. We observed that [(14)C]8-oxodG at medium concentrations of up to 2 pmol/ml was taken up by MCF-7 cells, phosphorylated to mono-, di-, and triphosphate derivatives, and incorporated into DNA. Oxidative stress caused by exposure of the cells to 17beta-estradiol resulted in a reduction in the rate of [(14)C]8-oxodG incorporation into DNA and an increase in the ratio of 8-oxodG monophosphate (8-oxodGMP) to 8-oxodG triphosphate (8-oxodGTP) in the nucleotide pool. 17beta-Estradiol-induced oxidative stress up-regulated the nucleotide pool cleansing enzyme MTH1 and possibly other Nudix-related pyrophosphohydrolases. These data support the conclusion that 8-oxodGTP is formed in the nucleotide pool by both 8-oxodG metabolism and endogenous reactive oxygen species. The metabolism of 8-oxodG to 8-oxodGTP, followed by incorporation into DNA is a mechanism by which the cellular presence of this oxidized nucleoside can lead to mutations.