Evaluation of effects of gamma-irradiation at low doses on repair and meiotic recombination mutants of Drosophila melanogaster

A comparative study of the effects of gene mutations mus209, mus309, mei-41 and rad54 of Drosophila melanogaster on the sensitivity to low-level exposure of different duration was carried out. Taken into account was the survival rate at different stages of ontogeny, female fecundity, the frequency of dominant lethal mutations (DLM) and the DNA damage. mei-41 and rad-54 mutants were most sensitive to the action of low dose radiation (80 mGy) in terms of survival and DLM. However, at the level of DNA damage, an increased radiosensitivity is observed only at larger doses of low intensity irradiation. Based on these observations, we can conclude about the importance of repair and its genes in the formation of the effect of low level doses of ionizing radiation in Drosophila.     

Postreplication repair defects in mutants of Drosophila melanogaster

Summary Mutants of Drosophila melanogaster which are defective in DNA synthesis have been identified among mutagen-sensitive stocks through analysis of both organ and cell cultures. A new procedure employing larval brain ganglia allows poorly fertile or sterile mutants to be analyzed for the first time. Parallel studies were performed in both tissues to establish the sensitivity of the new assay relative to that of the proven cell-culture assay. Damage was induced in the DNA of cultured cells with UV irradiation and in that of ganglial cells with the carcinogen N-acetoxy-2-acetylaminofluorene. Cultures were then pulse-labeled with ³H-thymidine, incubated in the absence of thymidine, and the newly synthesized DNA was analyzed by alkaline sucrose gradient centrifugation. The molecular weight of labeled DNA from mutant cells was compared with that from control cells to assess the effect of the mutant on DNA synthesis. Among 16 mutant stocks that were scanned in either or both tissues, seven show reductions in DNA synthesis using an undamaged template. Mutants at five different genetic loci [mus(2)205, mus(3)304, mus(3)308, mus(3)310 and mus(3)311] possess a reduced capacity to synthesize DNA on a UV-damaged template in primary cell cultures. Four of these five defects can also be detected in carcinogen-treated organ cultures. Two additional defects in postreplication repair were observed with the brainganglia assay in strains that cannot be assayed in cell culture [mus(1)108, mus(2)206].Abbreviations MMS methyl methanesulfonate - HN2 nitrogen mustard - AAF 2-acetylaminofluorene - AAAF N-acetoxy-2-acetylaminofluorene - DMSO dimethyl sulfoxide     

DNA double-strand damage and repair following gamma-irradiation in isolated spermatogenic cells

Various cell types in spermatogenesis exhibit differential sensitivity to radiation-induced DNA damage. The investigation of DNA radiosensitivity in vitro is complicated by the heterogeneous population of male germ cells (MGC) present in isolated single-cell suspensions. In the present investigation, the neutral elution technique was used to assess gamma-irradiation-induced DNA double-strand damage (DSD) in spermatogonia and preleptotene spermatocytes (SG/PL), pachytene spermatocytes and spermatid spermatocytes, as well as in MGC. In addition, the capability of these cell types to repair DNA double-strand damage was investigated. Based on the well established timing of the rat spermatogenic cycle, the DNA of specific cell populations was labeled using tritiated thymidine. DNA from labeled cells was determined isotopically, whereas total DNA was quantitated using a fluorometric method. DSD was induced in a dose-dependent manner in the heterogeneous population as well as in the labeled cell populations. SG/PL were more sensitive to gamma-irradiation-induced DSD than either the heterogeneous MGC population, pachytene or spermatid spermatocytes. Each cell type exhibited a similar capability to repair DSD following exposure to 3000 rad; repair was rapid (maximal within 45 min) and incomplete (less than 40%). Only pachytene spermatocytes exhibited significant repair following exposure to 6000 rad. Since a difference in sensitivity to radiation-induced DSD was demonstrated, the capability of each cell type to repair a similar initial frequency of strand damage was investigated. SG/PL, pachytene and spermatid spermatocytes differed in their capability to repair similar levels of strand damage. However, the difference in dose required to achieve equal damage may have contributed to other cellular effects, thus altering repair. In summary, a model is described that permits the evaluation of genotoxic responses in specific populations of spermatogenic cells within a heterogeneous cell suspension. The ability of specific cell types to repair gamma-irradiation-induced DNA double-strand damage is demonstrated.     

The multifaceted influence of histone deacetylases on DNA damage signalling and DNA repair

Histone/protein deacetylases play multiple roles in regulating gene expression and protein activation and stability. Their deregulation during cancer initiation and progression cause resistance to therapy. Here, we review the role of histone deacetylases (HDACs) and the NAD⁺ dependent sirtuins (SIRTs) in the DNA damage response (DDR). These lysine deacetylases contribute to DNA repair by base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), homologous recombination (HR) and interstrand crosslink (ICL) repair. Furthermore, we discuss possible mechanisms whereby these histone/protein deacetylases facilitate the switch between DNA double-strand break (DSB) repair pathways, how SIRTs play a central role in the crosstalk between DNA repair and cell death pathways due to their dependence on NAD⁺, and the influence of small molecule HDAC inhibitors (HDACi) on cancer cell resistance to genotoxin based therapies. Throughout the review, we endeavor to identify the specific HDAC targeted by HDACi leading to therapy sensitization.     

Lethality of visible light for Escherichia coli hemH1 mutants influence of defects in DNA repair

The hemH gene encodes ferrochelatase, the final enzyme of the heme biosynthetic pathway. Defects of this enzyme lead to accumulation of protoporphyrin IX and an increase in reactive oxygen species, causing susceptibility to blue and white light in bacteria and protoporphyria in humans. Here we show that the photosensitivity of hemH1 strains is much increased when the bacteria are devoid of ability to repair abasic sites. The sensitivity is increased 10- or 50-fold, in mutants bearing single xth or triple xth-nth-nfo mutations, respectively, but is not changed in mutants bearing nth, fpg, mutY, and mutT that are positive or negative for uvrA. This may indicate that in hemH1 mutants abasic sites are accumulated to a greater degree than oxidised bases, and/or that protoporphyrin, in the presence of abasic sites, increases the photosensitivity of hemH1 cells. It was shown in this work that the level of abasic sites (and/or strand breaks) in DNA of hemH1 strains increases greatly. Abasic sites and oxidative bases are potential mutagenic lesions. Nevertheless, the sensitivity of hemH1 bacteria to the lethal effect of visible light is not accompanied by increase in mutations. One of the possible explanations is that the genotoxic effect due to damage of hemH, shortage of heme and/or accumulating of protoporphyrin IX makes mutagenesis impossible.     

The effects of dietary coenzyme Q on Drosophila life span

Reactive oxygen species (ROS), generated as by-products of aerobic metabolism, cause damage to proteins and cellular membranes, and are thus thought to influence senescence. Caenorhabditis elegans fed on diets lacking in ubiquinone coenzyme Q (CoQ), a coenzyme in the oxidative phosphorylation pathway, show increased longevity, possibly because of reduced ROS generation. We test the role of dietary CoQ in determining Drosophila melanogaster longevity by measuring survival and cytochrome c-oxidase activity (a proxy for aerobic metabolic performance) in flies fed wild-type yeast, CoQ-less yeast, or respiratory control (RC) yeast replete with CoQ but independently deficient in mitochondrial respiration. We find no evidence that dietary manipulation of CoQ in D. melanogaster increases life span or decreases age-dependent decline in cytochrome c oxidase activity. Instead, we find evidence that flies fed a diet of respiratory-deficient yeast (CoQ-less or RC) tend to have decreased longevity and increased rates of decline in cytochrome c-oxidase activity [corrected]     

DNA damage responses in Drosophila nbs mutants with reduced or altered NBS function

The MRN complex, composed of MRE11, RAD50 and NBS, plays important roles in responding to DNA double-strand breaks (DSBs). In metazoans, functional studies of genes encoding these proteins have been challenging because complete loss-of-function mutations are lethal at the organismal level and because NBS has multiple functions in DNA damage responses. To study functions of Drosophila NBS in DNA damage responses, we used a separation-of-function mutation that causes loss of the forkhead-associated (FHA) domain. Loss of the FHA domain resulted in hypersensitivity to ionizing radiation and defects in gap repair by homologous recombination, but had only a small effect on the DNA damage checkpoint response and did not impair DSB repair by end joining. We also found that heterozygosity for an nbs null mutation caused reduced gap repair and loss of the checkpoint response to low-dose irradiation. These findings shed light on possible sources of the cancer predisposition found in human carriers of NBN mutations.     

Null mutations in Drosophila N-acetylglucosaminyltransferase I produce defects in locomotion and a reduced life span

UDP-GlcNAc:alpha3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (encoded by Mgat1) controls the synthesis of hybrid, complex, and paucimannose N-glycans. Mice make hybrid and complex N-glycans but little or no paucimannose N-glycans. In contrast, Drosophila melanogaster and Caenorhabditis elegans make paucimannose N-glycans but little or no hybrid or complex N-glycans. To determine the functional requirement for beta1,2-N-acetylglucosaminyltransferase I in Drosophila, we generated null mutations by imprecise excision of a nearby transposable element. Extracts from Mgat1(1)/Mgat1(1) null mutants showed no beta1,2-N-acetylglucosaminyltransferase I enzyme activity. Moreover, mass spectrometric analysis of these extracts showed dramatic changes in N-glycans compatible with lack of beta1,2-N-acetylglucosaminyltransferase I activity. Interestingly, Mgat1(1)/Mgat1(1) null mutants are viable but exhibit pronounced defects in adult locomotory activity when compared with Mgat1(1)/CyO-GFP heterozygotes or wild type flies. In addition, in null mutants males are sterile and have a severely reduced mean and maximum life span. Microscopic examination of mutant adult fly brains showed the presence of fused beta lobes. The removal of both maternal and zygotic Mgat1 also gave rise to embryos that no longer express the horseradish peroxidase antigen within the central nervous system. Taken together, the data indicate that beta1,2-N-acetylglucosaminyltransferase I-dependent N-glycans are required for locomotory activity, life span, and brain development in Drosophila.     

X-ray sensitivity and single-strand DNA break repair in mutagen-sensitive mutants of Drosophila melanogaster

Mutants of Drosophila melanogaster that are sensitive to chemical mutagens were analyzed for sensitivity to X-rays and for the capacity to repair single-strand DNA breaks induced by X-rays. Analysis of X-ray sensitivity demonstrated that 74% of the mutants assayed display some X-ray sensitivity, with 75% of the sensitive lines being extremely sensitive. Repair of single-strand breaks was assayed after both high and low doses of irradiation in order to permit detection of repair over a wide range of damage. The results of this investigation fail to show a correlation between X-ray sensitivity and this particular repair process. Repair of single-strand breaks is therefore mediated by repair processes unrelated to those that are disrupted in the current mutant collection.     

Effect of gamma-irradiation on oogenesis of Drosophila mutants defective for reparation and meiotic recombination

Effects of mutations rad201, mei-9, and mei-41 on cell sensitivity to gamma-radiation in Drosophila oogenesis were studied. Females of the control (Oregon R) and mutant strains were irradiated at a dose of 15 Gy. For 9 days after the irradiation, the number of eggs in consecutive day batches, the frequency of dominant lethals (DLs) among the eggs, and the cytologically recorded distribution of oocytes for stages of their development, and the frequency of egg chamber degeneration in female ovaries were estimated. As a result of joint analysis of the data, different oogenesis stages were characterized with regard to the frequency of two radiation-induced events: appearance of DLs in oocytes and degeneration of egg chambers due to apoptosis of nurse cells. It was shown that the mutations affect these parameters only at particular stages of early oogenesis, at which previtellogenetic growth of egg follicles and meiotic recombination in oocytes occur. Mutation rad201G1 increased the frequency of DLs and egg chamber degeneration, mei-41D5 affected only the DL frequency, and mei-9a, in addition to enhancing the chamber degeneration frequency, promoted radiation "rescue" of some oocytes from the DL induction.     

Shortening of the life span in an experiment with chronic external gamma-irradiation. The structure of mortality

The paper submits the statistical analysis of data on the mortality structure obtained by the experiment with chronic external gamma-irradiation within a wide range of dose rates. The cumulative mortality rate was a function of time. The authors discuss whether the shortening of life span resulting from chronic exposure to gamma-radiation is or is not specific.     

Effects of mutations in DNA repair genes on formation of ribosomal DNA circles and life span in Saccharomyces cerevisiae

A cause of aging in Saccharomyces cerevisiae is the accumulation of extrachromosomal ribosomal DNA circles (ERCs). Introduction of an ERC into young mother cells shortens life span and accelerates the onset of age-associated sterility. It is important to understand the process by which ERCs are generated. Here, we demonstrate that homologous recombination is necessary for ERC formation. rad52 mutant cells, defective in DNA repair through homologous recombination, do not accumulate ERCs with age, and mutations in other genes of the RAD52 class have varying effects on ERC formation. rad52 mutation leads to a progressive delocalization of Sir3p from telomeres to other nuclear sites with age and, surprisingly, shortens life span. We speculate that spontaneous DNA damage, perhaps double-strand breaks, causes lethality in mutants of the RAD52 class and may be an initial step of aging in wild-type cells.     

The repair of G-quadruplex-induced DNA damage

G4 DNA motifs, which can form stable secondary structures called G-quadruplexes, are ubiquitous in eukaryotic genomes, and have been shown to cause genomic instability. Specialized helicases that unwind G-quadruplexes in vitro have been identified, and they have been shown to prevent genetic instability in vivo. In the absence of these helicases, G-quadruplexes can persist and cause replication fork stalling and collapse. Translesion synthesis (TLS) and homologous recombination (HR) have been proposed to play a role in the repair of this damage, but recently it was found in the nematode Caenorhabditis elegans that G4-induced genome alterations are generated by an error-prone repair mechanism that is dependent on the A-family polymerase Theta (Pol θ). Current data point towards a scenario where DNA replication blocked at G-quadruplexes causes DNA double strand breaks (DSBs), and where the choice of repair pathway that can act on these breaks dictates the nature of genomic alterations that are observed in various organisms.     

Lipid peroxidation and the life span of Drosophila melanogaster

Studies have been made on the relationship between incubation temperature (20-30 degrees C) of D. melanogaster and the life span as well as the content of various products of lipid peroxidation. It was shown that the increase in the environmental temperature results in the decrease in the life span, the content of unsaturated fatty acids and conjugated hydroxyperoxids; ketodienic content increases. Strong correlation was observed between the life span and the content of peroxidation products. As it is indicated by coefficients of bifactorial linear regression with interaction, conjugated hydroperoxids and ketodiens exert negative influence on the life span. Their combined effect on the life span is less significant than the sum of their separate effects, which indicates the existence of common "canals" of their influences on the life span.     

Differential response to UV stress and DNA damage during the yeast replicative life span

The yeast Saccharomyces cerevisiae is mortal. Before they die, individual yeasts bud repeatedly producing a finite number of progeny, which have the capacity for a full life span. A feature of aging in many species is the waning of resistance to stress. To determine whether this is the case in yeast, we have examined the survival (viability) of age-synchronized populations of yeasts of various ages, spanning youth, midlife, and old age, after irradiation with ultraviolet light (UV). Resistance to UV was biphasic. There was an increase through midlife, followed by a precipitous decline. For comparison, another mutagenic agent, ethyl methanesulfonate (EMS), was tested in the same way. The response was very different. A uniphase decrease in resistance to this DNA-alkylating agent was found with a plateau later in life. The results argue that the increase in resistance to UV with age is an active process and not simply a monotonic age change. RAS2 is among the genes that determine yeast longevity. This gene is preferentially expressed in young cells and has a life span-extending effect on yeasts. One known function of RAS2 is to mount a protective response to irradiation by UV, which occurs independently of DNA damage. The distinction between UV and EMS found here is consistent with the notion that resistance to UV plays a role in yeast longevity in a manner not related to DNA damage. Furthermore, it suggests that RAS2 may participate in this response. We have found that RAS2 expression and UV resistance coincide in middle-aged yeasts bolstering this possibility. These data and the eclipse in activity of several longevity determining genes at midlife in yeasts also raise the possibility that active life maintenance processes function through this period, after which the organism operates on any remaining reserves until death. © 1996 Wiley-Liss, Inc.     

DNA repair defects and other (mus)takes in Drosophila melanogaster.

Preservation of the structural integrity of DNA in any organism is crucial to its health and survival. Such preservation is achieved by an extraordinary cellular arsenal of damage surveillance and repair functions, many of which are now being defined at the gene and protein levels. Mutants hypersensitive to the killing effects of DNA-damaging agents have been instrumental in helping to identify DNA repair-related genes and to elucidate repair mechanisms. In Drosophila melanogaster, such strains are generally referred to as mutagen-sensitive (mus) mutants and currently define more than 30 genetic loci. Whereas most mus mutants have been recovered on the basis of hypersensitivity to the monofunctional alkylating agent methyl methanesulfonate, they nevertheless constitute a phenotypically diverse group, with many mutants having effects beyond mutagen sensitivity. These phenotypes include meiotic dysfunctions, somatic chromosome instabilities, chromatin abnormalities, and cell proliferation defects. Within the last few years numerous mus and other DNA repair-related genes of Drosophila have been molecularly cloned, providing new insights into the functions of these genes. This article outlines strategies for isolating mus mutations and reviews recent advances in the Drosophila DNA repair field, emphasizing mutant analysis and gene cloning.     

Extension of Drosophila melanogaster life span with a GPCR peptide inhibitor

G protein-coupled receptors (GPCRs) mediate signaling from extracellular ligands to intracellular signal transduction proteins. Methuselah (Mth) is a class B (secretin-like) GPCR, a family typified by their large, ligand-binding, N-terminal extracellular domains. Downregulation of mth increases the life span of Drosophila melanogaster; inhibitors of Mth signaling should therefore enhance longevity. We used mRNA display selection to identify high-affinity (K(d) = 15 to 30 nM) peptide ligands that bind to the N-terminal ectodomain of Mth. The selected peptides are potent antagonists of Mth signaling, and structural studies suggest that they perturb the interface between the Mth ecto- and transmembrane domains. Flies constitutively expressing a Mth antagonist peptide have a robust life span extension, which suggests that the peptides inhibit Mth signaling in vivo. Our work thus provides new life span-extending ligands for a metazoan and a general approach for the design of modulators of this important class of GPCRs.     

Genetic modifiers of the Drosophila blue cheese gene link defects in lysosomal transport with decreased life span and altered ubiquitinated-protein profiles

Defects in lysosomal trafficking pathways lead to decreased cell viability and are associated with progressive disorders in humans. Previously we have found that loss-of-function (LOF) mutations in the Drosophila gene blue cheese (bchs) lead to reduced adult life span, increased neuronal death, and widespread CNS degeneration that is associated with the formation of ubiquitinated-protein aggregates. To identify potential genes that participate in the bchs functional pathway, we conducted a genetic modifier screen based on alterations of an eye phenotype that arises from high-level overexpression of Bchs. We found that mutations in select autophagic and endocytic trafficking genes, defects in cytoskeletal and motor proteins, as well as mutations in the SUMO and ubiquitin signaling pathways behave as modifiers of the Bchs gain-of-function (GOF) eye phenotype. Individual mutant alleles that produced viable adults were further examined for bchs-like phenotypes. Mutations in several lysosomal trafficking genes resulted in significantly decreased adult life spans and several mutants showed changes in ubiquitinated protein profiles as young adults. This work represents a novel approach to examine the role that lysosomal transport and function have on adult viability. The genes characterized in this study have direct human homologs, suggesting that similar defects in lysosomal transport may play a role in human health and age-related processes.