ATM- and ATR-mediated phosphorylation of XRCC3 regulates DNA double-strand break-induced checkpoint activation and repair

The RAD51 paralogs XRCC3 and RAD51C have been implicated in homologous recombination (HR) and DNA damage responses. However, the molecular mechanism(s) by which these paralogs regulate HR and DNA damage signaling remains obscure. Here, we show that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We find that RAD51C but not XRCC2 is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G(2) phases. XRCC3 phosphorylation is required for chromatin loading of RAD51 and HR-mediated repair of double-strand breaks (DSBs). Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G(2)/M checkpoint independently of its phosphorylation. Strikingly, we find that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation is required for the HR-mediated recovery of collapsed replication forks but is dispensable for the restart of stalled replication forks. Together, these findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair.

In Vivo Genotoxicity Assessment of Titanium Dioxide Nanoparticles by Allium cepa Root Tip Assay at High Exposure Concentrations

The industrial production and commercial applications of titanium dioxide nanoparticles have increased considerably in recent times, which has increased the probability of environmental contamination with these agents and their adverse effects on living systems. This study was designed to assess the genotoxicity potential of TiO2 NPs at high exposure concentrations, its bio-uptake, and the oxidative stress it generated, a recognised cause of genotoxicity. Allium cepa root tips were treated with TiO2 NP dispersions at four different concentrations (12.5, 25, 50, 100 mu g/mL). A dose dependant decrease in the mitotic index (69 to 21) and an increase in the number of distinctive chromosomal aberrations were observed. Optical, fluorescence and confocal laser scanning microscopy revealed chromosomal aberrations, including chromosomal breaks and sticky, multipolar, and laggard chromosomes, and micronucleus formation. The chromosomal aberrations and DNA damage were also validated by the comet assay. The bio-uptake of TiO2 in particulate form was the key cause of reactive oxygen species generation, which in turn was probably the cause of the DNA aberrations and genotoxicity observed in this study.

Toxische Wirkung von Schadstoffen auf Fischzellen. Bestimmung von DNA-Strangbrüchen mit dem Comet-Assay

The alkaline comet assay is a method of detecting DNA strand breaks and alkali labile sites in individual cells. The method was used to detect DNA strand breaks in isolated blood cells (leukocytes) of carp (Cyprius carpio). DNA damage have been induced by exposure of the cells to sediment extract. Therefore comet assay can be applied as in vitro bioassay for investigations on toxicity of marine sediments.

Expanding the repertoire of DNA-mediated signaling in DNA repair

DNA damage is extremely detrimental to the cell and must be repaired to protect the genome. DNA is capable of conducting charge through the overlapping π-orbitals of stacked bases; this phenomenon is extremely sensitive to the integrity of the π-stack, as perturbations attenuate DNA charge transport (CT). Based on the E. coli base excision repair (BER) proteins EndoIII and MutY, it has recently been proposed that redox-active proteins containing metal clusters can utilize DNA CT to signal one another to locate sites of DNA damage.

To expand our repertoire of proteins that utilize DNA-mediated signaling, we measured the DNA-bound redox potential of the nucleotide excision repair (NER) helicase XPD from Sulfolobus acidocaldarius. A midpoint potential of 82 mV versus NHE was observed, resembling that of the previously reported BER proteins. The redox signal increases in intensity with ATP hydrolysis in only the WT protein and mutants that maintain ATPase activity and not for ATPase-deficient mutants. The signal increase correlates directly with ATP activity, suggesting that DNA-mediated signaling may play a general role in protein signaling. Several mutations in human XPD that lead to XP-related diseases have been identified; using SaXPD, we explored how these mutations, which are conserved in the thermophile, affect protein electrochemistry.

To further understand the electrochemical signaling of XPD, we studied the yeast S. cerevisiae Rad3 protein. ScRad3 mutants were incubated on a DNA-modified electrode and exhibited a similar redox potential to SaXPD. We developed a haploid strain of S. cerevisiae that allowed for easy manipulation of Rad3. In a survival assay, the ATPase- and helicase-deficient mutants show little survival, while the two disease-related mutants exhibit survival similar to WT. When both a WT and G47R (ATPase/helicase deficient) strain were challenged with different DNA damaging agents, both exhibited comparable survival in the presence of hydroxyurea, while with methyl methanesulfonate and camptothecin, the G47R strain exhibits a significant change in growth, suggesting that Rad3 is involved in repairing damage beyond traditional NER substrates. Together, these data expand our understanding of redox-active proteins at the interface of DNA repair.

DNA-mediated charge transport signaling within the cell

DNA possesses the curious ability to conduct charge longitudinally through the π-stacked base pairs that reside within the interior of the double helix. The rate of charge transport (CT) through DNA has a shallow distance dependence. DNA CT can occur over at least 34 nm, a very long molecular distance. Lastly, DNA CT is exquisitely sensitive to disruptions, such as DNA damage, that affect the dynamics of base-pair stacking. Many DNA repair and DNA-processing enzymes are being found to contain 4Fe-4S clusters. These co-factors have been found in glycosylases, helicases, helicase-nucleases, and even enzymes such as DNA polymerase, RNA polymerase, and primase across the phylogeny. The role of these clusters in these enzymes has remained elusive. Generally, iron-sulfur clusters serve redox roles in nature since, formally, the cluster can exist in multiple oxidation states that can be accessed within a biological context. Taken together, these facts were used as a foundation for the hypothesis that DNA-binding proteins with 4Fe-4S clusters utilize DNA-mediated CT as a means to signal one another to scan the genome as a first step in locating the subtle damage that occurs within a sea of undamaged bases within cells.

Herein we describe a role for 4Fe-4S clusters in DNA-mediated charge transport signaling among EndoIII, MutY, and DinG, which are from distinct repair pathways in E. coli. The DinG helicase is an ATP-dependent helicase that contains a 4Fe-4S cluster. To study the DNA-bound redox properties of DinG, DNA-modified electrochemistry was used to show that the 4Fe-4S cluster of DNA-bound DinG is redox-active at cellular potentials, and shares the 80 mV vs. NHE redox potential of EndoIII and MutY. ATP hydrolysis by DinG increases the DNA-mediated redox signal observed electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.

In separate work presented here, it is shown that the UvrC protein from E. coli contains a hitherto undiscovered 4Fe-4S cluster. A broad shoulder at 410 nm, characteristic of 4Fe-4S clusters, is observed in the UV-visible absorbance spectrum of UvrC. Electron paramagnetic resonance spectroscopy of UvrC incubated with sodium dithionite, reveals a spectrum with the signature features of a reduced, [4Fe-4S]+1, cluster. DNA-modified electrodes were used to show that UvrC has the same DNA-bound redox potential, of ~80 mV vs. NHE, as EndoIII, DinG, and MutY. Again, this means that these proteins are capable of performing inter-protein electron transfer reactions. Does UvrC use DNA-mediated signaling to facilitate the repair of its substrates?

UvrC is part of the nucleotide excision repair (NER) pathway in E. coli and is the protein within the pathway that performs the chemistry required to repair bulky DNA lesions, such as cyclopyrimidine dimers, that form as a product of UV irradiation. We tested if UvrC utilizes DNA-mediated signaling to facilitate the efficient repair of UV-induced DNA damage products by helping UvrC locate DNA damage. The UV sensitivity of E. coli cells lacking DinG, a putative signaling partner of UvrC, was examined. Knocking out DinG in E. coli leads to a sensitivity of the cells to UV irradiation. A 5-10 fold reduction in the amount of cells that survive after irradiation with 90 J/m2 of UV light is observed. This is consistent with the hypothesis that UvrC and DinG are signaling partners, but is this signaling due to DNA-mediated CT? Complementing the knockout cells with EndoIII D138A, which can also serve as a DNA CT signaling partner, rescues cells lacking DinG from UV irradiation, while complementing the cells with EndoIII Y82A shows no rescue of viability. These results indicate that there is cross-talk between the NER pathway and DinG via DNA-mediated signaling. Perhaps more importantly, this work also establishes that DinG, EndoIII, MutY, and UvrC comprise a signaling network that seems to be unified by the ability of these proteins to perform long range DNA-mediated CT signaling via their 4Fe-4S clusters.

Involvement of ANXA5 and ILKAP in Susceptibility to Malignant Melanoma

Single nucleotide-polymorphisms (SNPs) are a source of diversity among human population, which may be responsible for the different individual susceptibility to diseases and/or response to drugs, among other phenotypic traits. Several low penetrance susceptibility genes associated with malignant melanoma (MM) have been described, including genes related to pigmentation, DNA damage repair and oxidative stress pathways. In the present work, we conducted a candidate gene association study based on proteins and genes whose expression we had detected altered in melanoma cell lines as compared to normal melanocytes. The result was the selection of 88 loci and 384 SNPs, of which 314 fulfilled our quality criteria for a case-control association study. The SNP rs6854854 in ANXA5 was statistically significant after conservative Bonferroni correction when 464 melanoma patients and 400 controls were analyzed in a discovery Phase I. However, this finding could not be replicated in the validation phase, perhaps because the minor allele frequency of SNP rs6854854 varies depending on the geographical region considered. Additionally, a second SNP (rs6431588) located on ILKAP was found to be associated with melanoma after considering a combined set of 1,883 MM cases and 1,358 disease-free controls. The OR was 1.29 (95% CI 1.12-1.48; p-value= 4x10(-4)). Both SNPs, rs6854854 in ANXA5 and rs6431588 in ILKAP, show population structure, which, assuming that the Spanish population is not significantly structured, suggests a role of these loci on a specific genetic adaptation to different environmental conditions. Furthermore, the biological relevance of these genes in MM is supported by in vitro experiments, which show a decrease in the transcription levels of ANXA5 and ILKAP in melanoma cells compared to normal melanocytes.

Efeito antimutagênico, antigenotóxico, antiobesidade e antioxidante da Ilex paraguariensis (erva-mate)

O extrato aquoso de erva-mate, obtido a partir de folhas secas de Ilex paraguariensis, é uma bebida amplamente consumida na América do Sul. Inicialmente, nosso objetivo foi caracterizar os compostos presentes nas amostras de erva-mate disponíveis no mercado brasileiro (CH: chimarrão; T: chá mate torrado; G: chá mate torrado, comercialmente acondicionado em garrafas ou C: em copos; TS: chá mate torrado solúvel A mutagenicidade, citotoxicidade e antimutagenicidade de todas as amostras também foram avaliadas atavés do Teste de Ames na presença e na ausência de ativação metabólica. Em seguida, analisamos a amostra TS (2,5, 5,0 e 10 mg/mL) quanto a sua atividade antioxidante e antigenotóxica. Além disso, avaliamos também os efeitos da amostra TS sobre a sinalização da leptina e da insulina no hipotálamo e o estresse oxidativo hepático de ratos adultos obesos programados pela superalimentação neonatal (S). Para induzir S, o tamanho da ninhada foi reduzido a três filhotes por lactante e as ninhadas com número padrão de filhotes (dez/lactante) foram utilizadas como controle. Aos 150 dias de vida, as proles S foram subdivididas em: TS - tratados com extrato aquoso de erva-mate (1g/kg de peso corporal/dia, por gavagem) e S - recebendo água por gavagem durante 30 dias. A prole controle (C) também recebeu água nas mesmas condições do grupo S. Em nossos resultados, verificamos a presença de ácido clorogênico, cafeína e teobromina em todas as amostras analisadas. O conteúdo de compostos fenólicos nas infusões estudadas foram CH: 5,140,23; T: 4,330,01; G: 0,930,25; C: 0,800,3 e TS: 8,350,5 mg/ml. Não observamos efeito mutagênico ou citotóxico nas amostras analisadas. Um efeito antimutagênico significativo foi observado para a cepa TA97 (pré-, co- e pós-tratamento), na presença de ativação metabólica, em todas as amostras testadas. A amostra TS também apresentou um efeito antimutagênico significativo para a TA102 (pré-, co-e e pós-tratamento), na presença de ativação metabólica. Na análise exclusiva da amostra TS, observamos uma atividade antioxidante quando utilizado o ensaio de DPPH, apresentando IC50 69,3+3,1 μg/ml. Além disso, a amostra TS apresentou um efeito protetor sobre a quebra do DNA plasmidial induzida por radicais superóxido e hidroxila, de maneira dose dependente. No teste do cometa, detectamos um efeito antigenotóxico induzido pelo TS em cultura primária de células epiteliais de esôfago. Em nossos testes in vivo observamos que os animais TS não desenvolveram sobrepeso, obesidade visceral e hiperfagia. A resistência hipotalâmica à leptina não foi significativamente revertida, porém a resistência à insulina foi minimizada pelo tratamento com TS no grupo programado pela S. No fígado, TS normalizou as atividades das enzimas antioxidantes (SOD, GPx e CAT) e diminuiu os marcadores de estresse oxidativo, MDA e 4-HNE. O tratamento com TS também reduziu o conteúdo de glicogênio e triglicerídios hepáticos. Nossos resultados sugerem que a erva-mate foi capaz de proteger o DNA contra danos oxidativos, aumentou as defesas antioxidantes, melhorou a função hepática em ratos superalimentados na lactação, talvez através da modulação da sinalização hipotalâmica da insulina podendo ser, portanto, uma importante ferramenta para prevenção e tratamento de doenças relacionadas ao estresse oxidativo.

Combined effects of ultraviolet radiation and temperature on morphology, photosynthesis, and DNA of Arthrospira (Spirulina) platensis (Cyanophyta)

Natural levels of solar UVR were shown to break and alter the spiral structure of Arthrospira (Spirulina) platensis (Nordst.) Gomont during winter. However, this phenomenon was not observed during summer at temperatures of similar to 30 degrees C. Since little has been documented on the interactive effects of solar UV radiation (UVR; 280-400 nm) and temperature on cyanobacteria, the morphology, photosynthesis, and DNA damage of A. platensis were examined using two radiation treatments (PAR [400-700 nm] and PAB [PAR + UV-A + UV-B: 280-700]), three temperatures (15, 22, and 30 degrees C), and three biomass concentrations (100, 160, and 240 mg dwt [dry weight] . L-1). UVR caused a breakage of the spiral structure at 15 degrees C and 22 degrees C, but not at 30 degrees C. High PAR levels also induced a significant breakage at 15 degrees C and 22 degrees C, but only at low biomass densities, and to lesser extent when compared with the PAB treatment. A. platensis was able to alter its spiral structure by increasing helix tightness at the highest temperature tested. The photochemical efficiency was depressed to undetectable levels at 15 degrees C but was relatively high at 30 degrees C even under the treatment with UVR in 8 h. At 30 degrees C, UVR led to 93%-97% less DNA damage when compared with 15 degrees C after 8 h of exposure. UV-absorbing compounds were determined as negligible at all light and temperature combinations. The possible mechanisms for the temperature-dependent effects of UVR on this organism are discussed in this paper.

Potential role of DNA-dependent protein kinase in cellular resistance to ionizing radiation

In this paper, we study the ability of DNA-PK-deficient (M059J) and -proficient (M059K) cells to undergo the rate of cellular proliferation, cell cycle distribution and apoptosis after 10 Gy X-ray irradiation, and the role of DNA-PK in radiosensitivity. The results showed that M059J cells exhibited hyper-radiosensitivity compared with M059K cells. A strong G2 phase arrest was observed in M059J cells post irradiation. Significant accumulation in the G2 phase in M059J cells was accompanied by apoptosis at 12 h. Altogether, the data suggested that DNA-PK may have two roles in mammalian cells after DNA damage, a role in DNA DSB repair and a second role in DNA-damaged cells to traverse a G2 checkpoint, by which DNA-PK may affect cellular sensitivity to ionizing radiation. 地址: [Li Ning; Zhang Hong; Wang Yanling; Hao Jifang] Chinese Acad Sci, Inst Modern Phys, Lanzhou 730000, Peoples R China; [Li Ning; Zhang Hong; Wang Yanling; Hao Jifang] Key Lab Heavy Ion Radiat Med Gansu Prov, Lanzhou 730000, Peoples R China; [Li Ning; Wang Yanling] Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China; [Wang Xiaohu] Gansu Tumor Hosp, Dept Radiotherapy, Lanzhou 730050, Peoples R China

DNA损伤检验点蛋白表达对肿瘤细胞辐射敏感性的影响

本论文应用X射线和12C6+离子对不同肿瘤细胞：HL-60、K562、SMMC-7721和HepG2进行辐照，用克隆形成率和四唑盐比色法(MTT)测定四种细胞的辐射敏感性；通过流式细胞术测定细胞周期分布、细胞凋亡、ATM和SMC1蛋白的表达变化。应用免疫细胞化学法与流式检测相结合研究了γ-H2AX蛋白表达的时间效应与剂量效应之间的关系。实验结果表明，四种细胞的辐射敏感性由强到弱依次为HL-60>K562>SMMC-7721>HepG2。即ATM表达量越低的细胞对辐射越敏感，周期阻滞水平越低，细胞凋亡越明显，但辐照后ATM蛋白的表达无显著增加，说明ATM的表达量和功能状态与细胞辐射敏感性有关，但其表达水平不能完全反映ATM蛋白激酶的活性。对ATM表达量差异最显著的HepG2和HL-60细胞来说,辐照前SMC1的表达水平与细胞S期的含量没有直接关系，辐照后SMC1蛋白的上调表达在S期阻滞修复中发挥明显的作用。辐照后1h,HL-60和HepG2细胞的H2AX磷酸化水平随吸收剂量的增加呈线性正相关,但曲线斜率与细胞辐射抗性的差异没有直接的联系。γ-H2AX的消失率与存活分数存在良好的相关性，HepG2细胞抗辐射能力强，这一时间短，HL-60细胞抗辐射能力弱，这一时间长。可以用γ-H2AX的消失速率来评估细胞的辐射敏感性。 以SMMC-7721细胞为同步化细胞模型发现，与其它同步化方法相比，步进电机旋转同步化培养法对细胞损伤最小，同步化效率最高，达到M期＞90%，GO/G1期＞80%，S期＞60%，G2/M>50%。同种射线辐照，GO/G1期SMMC-7721细胞的存活相对G2/M期来说较高。12C6+离子辐照明显减小了二者敏感度的差异性

12C6+束和Mitomycin C 诱导的DNA损伤效应

本文旨在分别研究重离子束及MMC在诱导细胞的DNA损伤效应中一些具体的分子机制，为治疗的进行以及相关辅助药物的开发提供理论依据。本文探索的重点有两个，第一个是重离子束辐射诱导的DNA损伤效应及p53在其中的激活，第二个是MMC诱导的DNA损伤效应及p53和BRCA1、H2AX等分子在其中的角色。 1. 12C6+离子束诱导HeLa细胞DNA损伤效应为了研究HeLa细胞经过12C6+ 束辐照之后的DNA损伤效应，及这个过程中p53激活的分子机制。我们运用中性单细胞电泳技术，检测了HeLa细胞经过4Gy 12C6+ 束辐照0h、3h、6h和12h之后DNA的损伤情况，以及0.5Gy、1Gy、2Gy和4Gy 12C6+ 束辐照0h后的DNA损伤情况。同时运用细胞生长实时监测仪监测了HeLa细胞在经过0Gy、0.5Gy和1Gy 12C6+ 束辐照之后的生长变化，并运用AO/EB双染检测了辐照24小时后的凋亡情况。另外，利用8mmol/L的caffeine（抑制ATM和ATR）和20μmol/L的wortmannin（抑制ATM和DNA-PK）处理HeLa细胞后再进行1Gy 12C6+ 束辐照，通过western blot检测p53的表达。结果显示，12C6+ 束辐照可造成HeLa细胞的DNA损伤，损伤随剂量升高而升高但随时间降低；并诱导HeLa细胞发生凋亡；而且辐照后p53表达升高，但经过caffeine或者wortmannin预先处理的细胞p53均没有显著升高。我们的结论是：12C6+ 束辐照可造成HeLa细胞的DNA损伤并诱导损伤修复及凋亡等效应，损伤效应相关的分子p53被激活，并且激活依赖于ATM。 2. MMC诱导的DNA损伤效应在这一部分研究中，首先，我们利用与上面相同的研究方法，探讨了p53在MMC诱导的DNA损伤效应中的激活情况，结果显示，MMC诱导的DNA损伤效应并不依赖于p53。另外，我们还探讨了， BRCA1在FANCD2的γ-H2AX依赖性转移中的作用。MMC可造成DNA的ICL（interstrand cross-link）损伤，ICL可通过FA(Fanconi Anemia)通路进行修复。FANCD2是FA通路的核心分子，在DNA产生ICL时被各种分子修饰然后转移到损伤部分，这个过程的涉及到ATR、γ-H2AX及BRCA1等，本文试图探讨BRCA1在其中的作用方式。研究中，我们监测了不同处理（包括对照、caffeine（可抑制ATR）、MMC及MMC ＋caffeine）的HCC1937(BRCA1缺陷型)和MCF7（BRCA1野生型）细胞的生长；并用Western blot检测MMC处理之后HCC1937细胞γ-H2AX的表达情况。结果表明，MMC和caffeine均可以抑制HCC1937的生长，但caffeine和MMC+caffeine的抑制效果是一样的；MMC和caffeine均可以抑制MCF7的生长，且MMC+caffeine处理比仅进行caffeine处理的抑制作用强；MMC处理之后，HCC1937的γ-H2AX表达显著升高。我们的结论是，在FANCD2的γ-H2AX依赖性转移中，H2AX的磷酸化并不依赖于BRCA1，不过，BRCA1和ATR应该参与一个相同的分子事件，可能是FANCD2的磷酸化。这个有待进一步的实验验证

重离子引起DNA损伤及其对细胞周期进程的影响

目的：木论文重点研究重离子不同剂量离子辐照后DNA损伤程度的变化，以及进而引起的细胞周期改变等现象。为重离子治癌的临床应用积累必要的基础数据。材料与方法：采用兰州重离子研究装置（HIRFL）加速的碳、氖等重离子辐照体外培养的贴璧肿瘤细胞，以单细胞电泳法（SCGE）检测DNA的损伤程度；以流式细胞技术（FCM）检测细胞的周期改变现象。结果：1．SMMC-7721月干癌细胞经重离子（氖、碳）辐照后，DNA损伤现象明显，表现为单细胞电泳中出现的普遍的拖尾现象（t-test，P<0.001，compared with control。2．80MeV／u 2ONe10＋辐照后立即检测发现：2Gy造成100％的细胞损伤：8Gy照射造成80％的细胞严重损伤：且彗尾拖尾长度随剂量增加早．指数关系增长，仔值为0.99058。3．辐照后12小时，若干不同剂量辐照的样品其彗尾长度趋于相同：如05协、Ic）和ZGy辐照样品的彗尾长度分别为132.3±12.8、132.9±9.5和133.1±11.7μm，24h，时为35.0±3.9、35.5±4.1和48.2±6.Oμm，这说明在一定剂量范围内（0.5-2Gy）的辐照下，随着修复时间的延长，细胞DNA的损伤程度将趋于相同。同时，细胞继续孵育12小时，对于0.55-2Gy辐照组来说DNA的损伤情况是24小时内操作最严重的。4．辐照后24小时，0.5-2Gy辐照组埙份明显修复，略高于对照，但是对于4Gy和SGy辐照组仍带有明显的损伤现魏。簇说眼熏离子辐照（>4Gy）所致DNA报伤的不足修复性。5，DNA的损伤将导致细胞通过一系列调节机制抑制细胞周期的进行，为DNA修复系统提供充足的时间进行DNA修复，从而造成明显的细胞周期阳．滞现琢，这在重离子辐照实验中同样得到证实，尤其是S期、G2／M期阻净带现象非常明显。

Regulation of DNA Double Strand Break Response

To ensure genomic integrity, dividing cells implement multiple checkpoint pathways during the course of the cell cycle. In response to DNA damage, cells may either halt the progression of the cycle (cell cycle arrest) or undergo apoptosis. This choice depends on the extent of damage and the cell's capacity for DNA repair. Cell cycle arrest induced by double-stranded DNA breaks relies on the activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell cycle effectors (e.g., Chk2 and p53) to inhibit cell cycle progression. ATM is an S/T-Q directed kinase that is critical for the cellular response to double-stranded DNA breaks. Following DNA damage, ATM is activated and recruited to sites of DNA damage by the MRN protein complex (Mre11-Rad50-Nbs1 proteins) where ATM phosphorylates multiple substrates to trigger a cell cycle arrest. In cancer cells, this regulation may be faulty and cell division may proceed even in the presence of damaged DNA. We show here that the RSK kinase, often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that RSK disrupts the binding of the MRN complex to DSB DNA. RSK can directly phosphorylate the Mre11 protein at Ser 676 both in vitro and in intact cells and can thereby inhibit loading of Mre11 onto DSB DNA. Accordingly, mutation of Ser 676 to Ala can reverse inhibition of the DSB response by RSK. Collectively, these data point to Mre11 as an important locus of RSK-mediated checkpoint inhibition acting upstream of ATM activation.

The phosphorylation of Mre11 on Ser 676 is antagonized by phosphatases. Here, we screened for phosphatases that target this site and identified PP5 as a candidate. This finding is consistent with the fact that PP5 is required for the ATM-mediated DNA damage response, indicating that PP5 may promote DSB-induced, ATM-dependent DNA damage response by targeting Mre11 upstream of ATM.

Nucleolar organization, ribosomal DNA array stability, and acrocentric chromosome integrity are linked to telomere function.

The short arms of the ten acrocentric human chromosomes share several repetitive DNAs, including ribosomal RNA genes (rDNA). The rDNA arrays correspond to nucleolar organizing regions that coalesce each cell cycle to form the nucleolus. Telomere disruption by expressing a mutant version of telomere binding protein TRF2 (dnTRF2) causes non-random acrocentric fusions, as well as large-scale nucleolar defects. The mechanisms responsible for acrocentric chromosome sensitivity to dysfunctional telomeres are unclear. In this study, we show that TRF2 normally associates with the nucleolus and rDNA. However, when telomeres are crippled by dnTRF2 or RNAi knockdown of TRF2, gross nucleolar and chromosomal changes occur. We used the controllable dnTRF2 system to precisely dissect the timing and progression of nucleolar and chromosomal instability induced by telomere dysfunction, demonstrating that nucleolar changes precede the DNA damage and morphological changes that occur at acrocentric short arms. The rDNA repeat arrays on the short arms decondense, and are coated by RNA polymerase I transcription binding factor UBF, physically linking acrocentrics to one another as they become fusogenic. These results highlight the importance of telomere function in nucleolar stability and structural integrity of acrocentric chromosomes, particularly the rDNA arrays. Telomeric stress is widely accepted to cause DNA damage at chromosome ends, but our findings suggest that it also disrupts chromosome structure beyond the telomere region, specifically within the rDNA arrays located on acrocentric chromosomes. These results have relevance for Robertsonian translocation formation in humans and mechanisms by which acrocentric-acrocentric fusions are promoted by DNA damage and repair.

Consequences of heat stress on reproduction of cattle

Heat stress represents one of the major environmental factors that adversely affect the reproductive performance of cattle. In this paper the behavioral adjustments, physical mechanisms and physiological responses to heat loss are described; bos indicus adaptive advantages with respect to bos Taurus, pathophysiology of heat stress and heat stress effects in animal reproduction, both the male and the female.