The Photorepair and Photoisomerization of DNA Lesions in Etiolated Cucumber Cotyledons after Irradiation by UV-B Depends on Wavelength

To examine the light-dependent repair of DNA photo-lesions,such as, cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyriinidonephotoproducts [(6-4)pho-toproducts], we incubated cucumber cotyledonsunder monochromatic light (325-500 nm) after they had been exposedto UV-B irradiation. Irradiation at wavelengths between 375and 425 nm was the most efficient for photo-repair of CPDs.The dependence on wavelength for the removal of (6-4)photoproductsdiffered from that for the photorepair of CPDs. The (6-4)photoproductswere most efficiently removed upon irradiation at 325 nm witha second peak at 400 to 425 nm. An immunological assay usingantibodies specific to Dewar photoproducts revealed that irradiationat 325 nm induced the photoisomerization of (6-4)photoproductsto Dewar photoproducts. Thus, the most efficient wavelengthfor the photorepair of (6-4)photoprod-ucts was between 400 and425 nm. It seems likely that processes similar to those involvedin photorepair of CPDs might be involved in the photorepairof (6-4)photoprod-ucts. (Received October 15, 1997; Accepted April 22, 1998)     

Light-dependent Induction of Polyunsaturated Fatty Acid Biosynthesis in Greening Cucumber Cotyledons

Greening cucumber (Cucumis sativus L.) cotyledons exhibited dramatic increases in the ability to desaturate exogenously added [1-¹⁴C]oleic acid and [1-¹⁴C]linoleic acid within 2 to 3 hours of illumination. These increases were effectively inhibited by 10 micrograms per milliliter cycloheximide. Oleate desaturation remained at a high level in constant light for 5 to 6 days after induction and then declined by about 50%; when returned to the dark, the tissue showed a sharp decrease in conversion of [¹⁴C]oleate to [¹⁴C]linoleate. Linoleate desaturation reached a maximum about 15 hours after induction and declined immediately thereafter while the tissue still was in the light; after induction had peaked return of the tissue to the dark showed a dramatic fall of linoleate desaturation. The changes in desaturation were correlated with the conversion of the principal fatty acid in the etiolated cotyledons, linoleate, to α-linolenate, and with the assembly of the chlorophyll-containing photosynthetic membranes. The incorporation of [1-¹⁴C]acetate into lipids showed no significant light stimulation. The role of light in the regulation of certain aspects of plant metabolism during development is discussed.     

细菌DNA损伤修复的诱导、调控及结局的研究进展

DNA损伤修复(SOS反应)是细菌适应环境、抵抗外界压力和修复自身损伤的重要机制.为了解SOS反应的过程,全面揭示细菌生存机制,本研究对DNA损伤修复的过程、调节及适应性变化进行文献综述.结果 表明,内源和外源的诸多压力都可以激活SOS反应,抗生素是激活该反应的主要因素.RecA在感知外界压力和系统启动过程中发挥重要作...     

Induction of 33-kD and 60-kD Peroxidases during Ethylene-Induced Senescence of Cucumber Cotyledons

Ethylene enhanced the senescence of cucumber (Cucumis sativus L. cv `Poinsett 76') cotyledons. The effect of 10 microliters per liter ethylene was inhibited by 1 millimolar silver thiosulfate, an inhibitor of ethylene action. An increase in proteins with molecular weights of 33 to 30 kilodaltons and lower molecular weights (25, 23, 20, 16, 12, and 10 kilodaltons) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after ethylene enhanced senescence. The measurement of DNase and RNase activity in gels indicated that these new proteins were not nucleases. Two proteins from ethylene-treated cotyledons were purified on the basis of their association with a red chromaphore and subsequently were identified as peroxidases. The molecular weights and isoelectric points (pI) of two of these peroxidases were 33 kilodaltons (cationic, pI = 8.9) and 60 kilodaltons (anionic, pI = 4.0). The observation that [³⁵S]Na₂SO₄ was incorporated into these proteins during ethylene-enhanced senescence suggests that these peroxidases represent newly synthesized proteins. Antibodies to the 33-kilodalton peroxidase precipitated two in vitro translation products from RNA isolated from ethylene-treated but not from control cucumber seedlings. This indicates that the increase in 33-kilodalton peroxidase activity represents de novo protein synthesis. Both forms of peroxidase degraded chlorophyll in vitro, which is consistent with the hypothesis that peroxidases have catabolic or scavenging functions in senescent tissues.     

Extremophilic Acinetobacter Strains from High-Altitude Lakes in Argentinean Puna: Remarkable UV-B Resistance and Efficient DNA Damage Repair

High-Altitude Andean Lakes (HAAL) of the South American Andes are almost unexplored ecosystems of shallow lakes. The HAAL are recognized by a remarkably high UV exposure, strong changes in temperature and salinity, and a high content of toxic elements, especially arsenic. Being exposed to remarkably extreme conditions, they have been classified as model systems for the study of life on other planets. Particularly, Acinetobacter strains isolated from the HAAL were studied for their survival competence under strong UV-B irradiation. Clinical isolates, Acinetobacter baumannii and Acinetobacter johnsonii, served as reference material. Whereas the reference strains rapidly lost viability under UV-B irradiation, most HAAL-derived strains readily survived this exposure and showed less change in cell number after the treatment. Controls for DNA repair activity, comparing dark repair (DR) or photo repair (PR), gave evidence for the involvement of photolyases in the DNA repair. Comparative measurements by HPLC-mass spectrometry detected the number of photoproducts: bipyrimidine dimers under both PR and DR treatments were more efficiently repaired in the HAAL strains (up to 85?% PR and 38?% DR) than in the controls (31?% PR and zero DR ability). Analysis of cosmid-cloned total genomic DNA from the most effective DNA-photorepair strain (Ver3) yielded a gene (HQ443199) encoding a protein with clear photolyase signatures belonging to class I CPD-photolyases. Despite the relatively low sequence similarity of 41?% between the enzymes from Ver3 and from E. coli (PDB 1DNPA), a model-building approach revealed a high structural homology to the CPD-photolyase of E. coli.     

DNA Damage and Repair in Eukaryotic Cells

DAMAGE IN DNA AFTER IRRADIATION CAN BE CLASSIFIED INTO FIVE KINDS: base damage, single-strand breaks, double-strand breaks, DNA-DNA cross-linking, and DNA-protein cross-linking. Of these, repair of base damage is the best understood. In eukaryotes, at least three repair systems are known that can deal with base damage: photoreactivation, excision repair, and post-replication repair. Photoreactivation is specific for UV-induced damage and occurs widely throughout the biosphere, although it seems to be absent from placental mammals. Excision repair is present in prokaryotes and in animals but does not seem to be present in plants. Post-replication repair is poorly understood. Recent reports indicate that growing points in mammalian DNA simply skip past UV-induced lesions, leaving gaps in newly made DNA that are subsequently filled in by de novo synthesis. Evidence that this concept is oversimplified or incorrect is presented.-Single-strand breaks are induced by ionizing radiation but most cells can rapidly repair most or all of them, even after supralethal doses. The chemistry of the fragments formed when breaks are induced by ionizing radiation is complex and poorly understood. Therefore, the intermediate steps in the repair of single-strand breaks are unknown. Double-strand breaks and the two kinds of cross-linking have been studied very little and almost nothing is known about their mechanisms for repair.-The role of mammalian DNA repair in mutations is not known. Although there is evidence that defective repair can lead to cancer and/or premature aging in humans, the relationship between the molecular defects and the diseased state remains obscure.     

A Fluorescent Probe to Measure DNA Damage and Repair

DNA damage and repair is a fundamental process that plays an important role in cancer treatment. Base excision repair (BER) is a major repair pathway that often leads to drug resistance in DNA-targeted cancer chemotherapy. In order to measure BER, we have developed a near infrared (NIR) fluorescent probe. This probe binds to a key intermediate, termed apurinic/apyrimidinic (AP) site, in the BER pathway where DNA damage and repair occurs. We have developed an assay to show the efficacy of the probe binding to AP sites and have shown that it can distinguish AP sites in DNA extract from chemotherapy treated cells. This probe has potential application in monitoring patient response to chemotherapy and evaluating new drugs in development.     

Sirtuin家族与DNA损伤修复

DNA损伤的发生与积累是造成细胞功能紊乱的根本原因,也是引起衰老与肿瘤等疾病发生的关键事件。为维持机体自身遗传物质的完整性与稳定性,生物体内拥有多种针对不同类型DNA损伤的修复方式。Sirtuin蛋白是一组NAD+依赖的、高度保守的组蛋白去乙酰化酶,可通过去乙酰化作用调节众多底物蛋白质的表达、活性与稳定性。 近来的研究显示,DNA损伤修复途径的多个关键蛋白质是Sirtuin的下游底物。Sirtuin蛋白通过调节同源重组修复、非同源末端修复、核苷酸切除修复等途径中的核心蛋白质参与修复包括双链断裂（double stranded breakes, DSBs）在内的多种DNA损伤类型,从而在维持基因组稳定性、寿命以及细胞能量代谢调节等一系列生物学作用中发挥至关重要的作用。本综述将介绍近年来Sirtuin与DNA损伤修复的研究进展。     

DNA损伤修复过程表观遗传学改变

多种化学、物理及生物因素可诱发细胞DNA损伤,损伤后DNA损伤位点被相关损伤感受器识别,激活相应的修复通路进行DNA修复。越来越多的证据表明DNA甲基化状态、蛋白翻译后修饰、染色质重塑、miRNA等修饰方式参与了DNA的损伤修复。文章通过不同损伤修复通路中这些修饰的特点,阐述表观遗传学改变在DNA损伤修复发展过程中的作用机制。     

Comparative DNA Damage and Repair in Echinoderm Coelomocytes Exposed to Genotoxicants

The capacity to withstand and repair DNA damage differs among species and plays a role in determining an organism''s resistance to genotoxicity, life history, and susceptibility to disease. Environmental stressors that affect organisms at the genetic level are of particular concern in ecotoxicology due to the potential for chronic effects and trans-generational impacts on populations. Echinoderms are valuable organisms to study the relationship between DNA repair and resistance to genotoxic stress due to their history and use as ecotoxicological models, little evidence of senescence, and few reported cases of neoplasia. Coelomocytes (immune cells) have been proposed to serve as sensitive bioindicators of environmental stress and are often used to assess genotoxicity; however, little is known about how coelomocytes from different echinoderm species respond to genotoxic stress. In this study, DNA damage was assessed (by Fast Micromethod) in coelomocytes of four echinoderm species (sea urchins Lytechinus variegatus, Echinometra lucunter lucunter, and Tripneustes ventricosus, and a sea cucumber Isostichopus badionotus) after acute exposure to H₂O₂ (0–100 mM) and UV-C (0–9999 J/m²), and DNA repair was analyzed over a 24-hour period of recovery. Results show that coelomocytes from all four echinoderm species have the capacity to repair both UV-C and H₂O₂-induced DNA damage; however, there were differences in repair capacity between species. At 24 hours following exposure to the highest concentration of H₂O₂ (100 mM) and highest dose of UV-C (9999 J/m²) cell viability remained high (>94.6±1.2%) but DNA repair ranged from 18.2±9.2% to 70.8±16.0% for H₂O₂ and 8.4±3.2% to 79.8±9.0% for UV-C exposure. Species-specific differences in genotoxic susceptibility and capacity for DNA repair are important to consider when evaluating ecogenotoxicological model organisms and assessing overall impacts of genotoxicants in the environment.     

离体培养黄瓜子叶诱导雌花的研究

采用添加Spd和IAA的MS培养基培养离体黄瓜子叶,研究了Spd和IAA对雌花诱导的协同作用,及昼夜温差、培养基中N素和pH值对雌花诱导的影响。结果表明,分别添加Spd、IAA时的雌花诱导率和单株雌花数偏低或为0,12 mg·L^-1 Spd与0.01mg·L^-1 IAA 配合时的诱导效果明显高于单独处理的,而对照组未见雌花,说明Spd和IAA对雌花诱导的协同作用显著。在0、2、6、10℃昼夜温差,60、70、80、90 mmol·L^-1的N素含量和pH 5.4、5.8、6.2、6.6的培养条件下,70 mmol·L^-1 N、6℃温差和pH 6.2时的雌花诱导效果较好,表明适当提高昼夜温差、培养基中N素和pH值有利于黄瓜子叶的雌花诱导。     

DNA Damage Signalling and Repair in Dictyostelium discoideum

Repair of DNA double strand breaks (DSBs) is critical for the maintenance of genome integrity. DNA DSBs can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). Whilst HR requires sequences homologous to thedamaged DNA template in order to facilitate repair, NHEJ occurs through recognition of DNA DSBs by a variety of proteins that process and rejoin DNA termini by direct ligation. Here we review two recent reports that NHEJ is conserved in the social amoebaDictyostelium discoideum. Certain components of the mammalian NHEJ pathway that are absent in genetically tractable organisms such as yeast are present in Dictyostelium and we discuss potential directions for future research, in addition to considering this organism as a genetic model system for the study of NHEJ in vivo.     

Loss of Urokinase Receptor Sensitizes Cells to DNA Damage and Delays DNA Repair

DNA damage induced by numerous exogenous or endogenous factors may have irreversible consequences on the cell leading to cell cycle arrest, senescence and cell death. The DNA damage response (DDR) is powerful signaling machinery triggered in response to DNA damage, to provide DNA damage recognition, signaling and repair. Most anticancer drugs induce DNA damage, and DNA repair in turn attenuates therapeutic efficiency of those drugs. Approaches delaying DNA repair are often used to increase efficiency of treatment. Recent data show that ubiquitin-proteasome system is essential for signaling and repair of DNA damage. However, mechanisms providing regulation of proteasome intracellular localization, activity, and recruitment to DNA damage sites are elusive. Even less investigated are the roles of extranuclear signaling proteins in these processes. In this study, we report the involvement of the serine protease urokinase-type plasminogen activator receptor (uPAR) in DDR-associated regulation of proteasome. We show that in vascular smooth muscle cells (VSMC) uPAR activates DNA single strand break repair signaling pathway. We provide evidence that uPAR is essential for functional assembly of the 26S proteasome. We further demonstrate that uPAR mediates DNA damage-induced phosphorylation, nuclear import, and recruitment of the regulatory subunit PSMD6 to proteasome. We found that deficiency of uPAR and PSMD6 delays DNA repair and leads to decreased cell survival. These data may offer new therapeutic approaches for diseases such as cancer, cardiovascular and neurodegenerative disorders.     

Accumulation of Pathogenesis-Related Proteins in Tobacco Leaves Irradiated with UV-B

Nicotiana tabacum L. (cv. Petit Havana SR1) were grown under ultraviolet-B (UV-B, 290–320 nm) irradiation, and soluble proteins were extracted from the leaves. Two-dimensional electrophoresis revealed that a minimum of 12 polypeptides were induced by UV-B. Polypeptides which were so abundant as to be detectable by Coomassie brilliant blue staining were then subjected to N-terminal amino acid sequence analyses. Two of the polypeptides were identified as a 23 kDa protein of PS II and 6 as a pathogenesis-related protein 5 (PR-5). Immunoblotting demonstrated that other PR proteins, PR-1 and PR-3 were also induced by UV-B. Salicylic acid (SA), which is an important component of signal transduction that leads to the expression of PR proteins and exhibition of acquired resistance to pathogens, increased in response to exposure to UV-B. In addition, the activity of phenylalanine ammonialyase, which catalyzes the synthesis from phenylalanine of trans-cinnamic acid, the endogenous precursor of SA, was transiently increased by UV-B irradiation. These results suggest that UV-B activates the signal transduction pathway, which is a common step in pathogen infection. Received 8 May 2000/ Accepted in revised form 29 August 2000     

DNA Damage Checkpoint, Damage Repair, and Genome Stability

Genomic DNA is under constant attack from both endogenous and exogenous sources of DNA damaging agents. Without proper care, the ensuing DNA damages would lead to alteration of genomic structure thus affecting the faithful transmission of genetic information. During the process of evolution, organisms have acquired a series of mechanisms responding to and repairing DNA damage, thus assuring the maintenance of genome stability and faithful transmission of genetic information. DNA damage checkpoint is one such important mechanism by which, in the face of DNA damage, a cell can respond to amplified damage signals, either by actively halting the cell cycle until it ensures that critical processes such as DNA replication or mitosis are complete or by initiating apoptosis as a last resort. Over the last decade, complex hierarchical interactions between the key components like ATM/ATR in the checkpoint pathway and various other mediators, effectors including DNA damage repair proteins have begun to emerge. In the meantime, an intimate relationship between mechanisms of damage checkpoint pathway, DNA damage repair, and genome stability was also uncovered. Reviewed hereinare the recent findings on both the mechanisms of activation of checkpoint pathways and their coordination with DNA damage repair machinery as well as their effect on genomic integrity.     

DNA损伤检验点与损伤修复及基因组稳定性

生物有机体基因组DNA经常会受到内源或外源因素的影响而导致结构发生变化,产生损伤;在长期进化过程中,有机体也相应形成了一系列应对与修复损伤DNA,并维持染色体基因组正常结构功能的机制。其中DNA损伤检验点（DNA damage checkpoint）就是在感应DNA损伤的基础上,对损伤感应信号进行转导,或引起细胞周期的暂停,从而使细胞有足够的时间对损伤DNA进行修复,或最终导致细胞发生凋亡。DNA损伤检验点信号转导途径是一个高度保守的信号感应过程,整个途径大致可以分为损伤感应、信号传递及信号效应3个组成部分。其中3-磷脂酰肌醇激酶家族类成员ATM（ataxia-telangiectasia mutated）和ATR（ataxia-telangiectasia and Rad3-related）活性的增加构成整个途径活化的第一步。它们通过激活下游的效应激酶,Chk2／Chk1,通过协同作用许多其他调控细胞周期、DNA复制、DNA损伤修复及细胞凋亡等过程的蛋白质因子来实现细胞对DNA损伤的高度协调反应。近十几年,随着此领域研究的不断深入,人们逐步揭示了DNA损伤检验点途径发生过程中,各种核心组分通过与不同调节因子、效应因子及DNA损伤修复蛋白间的复杂相互作用,以实现监测感应异常DNA结构并实施相应反应的机制;其中,检验点衔接因子（mediators）及染色质结构,尤其是核小体组蛋白的共价修饰在调控ATM／ATR活性,促进ATM／ATR与底物间的相互作用以及介导DNA损伤位点周围染色质区域上多蛋白复合物在时间与空间上的动态形成发挥着重要的作用。同时,人们也开始发现DNA损伤检验点途径与DNA损伤修复、基因组稳定性以及肿瘤发生等过程之间某些内在的联系。该反应途径在通过协调细胞针对DNA损伤做出各种反应的基础上,直接或间接地参与或调控DNA损伤修复过程,并与DNA损伤修复途径协同作用最终保证染色体基凶组结构的完整性,而检验点途径的改变,则会引起基因组不稳定的发生,包括从突变频率的提高到大范围的染色体重排,以及染色体数量的畸变。如：突变发生在肿瘤形成早期,会大大增加肿瘤发生的几率。文章将对DNA损伤检验点途径机制及其对DNA损伤修复、基因组稳定性影响的最新进展进行综述。     

DNA Damage Tolerance and a Web of Connections with DNA Repair at Yale

This short article summarizes some of the research carried out recently by my laboratory colleagues on the function of DNA polymerase zeta (polζ) in mammalian cells. Some personal background is also described, relevant to research associations with Yale University and its continuing influence. Polζ is involved in the bypass of many DNA lesions by translesion DNA synthesis and is responsible for the majority of DNA damage-induced point mutagenesis in mammalian cells (including human cells), as well as in yeast. We also found that the absence of this enzyme leads to gross chromosomal instability in mammalian cells and increased spontaneous tumorigenesis in mice. Recently, we discovered a further unexpectedly critical role for polζ: it plays an essential role in allowing continued rapid proliferation of cells and tissues. These observations and others indicate that polζ engages frequently during DNA replication to bypass and tolerate DNA lesions or unusual DNA structures that are barriers for the normal DNA replication machinery.