Evidence for nucleus independent steps in control of repair of mitochondrial damage

Summary The induction of the cytoplasmic petite mutation by ultraviolet light in 21 UV-sensitive (rad) nuclear mutants of Saccharomyces cerevisiae was compared to that in the wild type. Six rad mutants showed an increased sensitivity and two were less sensitive than the wild type. Modifications in the dose-response paralleled that of UV-induced reversion in one nuclear locus (hi ₁) studied. In these eight mutants the repair of UV-induced mitochondrial lesions seems to be under nuclear control.A block in the repair steps controlled by eleven of the other rad genes studied did not interfere with the repair of mitochondrial damage. In strains carrying a mutation in any one of these genes the dose-response curve for petite induction could be superimposed on that of the wild type even though they differed from the wild type in respect of nuclear gene reversion. These steps of the mitochondrial repair pathway(s) are therefore likely to be controlled by a nuclear and/or a cytoplasmic genetic determinant whose product acts specifically on mitochondrial lesions or it may be that the products of these genes are not required in the process of induction of petites.     

Contribution of a caffeine-sensitive recombinational repair pathway to survival and mutagenesis in UV-irradiated Schizosaccharomyces pombe

Summary Cells of wild-type Schizosacharomyces pombe exposed to UV radiation in either G1 or G2 phase show enhanced inactivation of colony-forming ability if plated in the presence of caffeine. This UV-sensitization by caffeine is abolished in both G1 and G2 phase cells by the rad1 mutation; since both caffeine and the rad1 mutation markedly reduce recombinational events, this suggests that a recombinational repair process is active in cells irradiated either in G1 or G2 phase. A prereplicative or sister chromatid exchange recombinational process appears to account for caffeine-sensitive repair of UV-damage in G2 cells (which possess at the time of radiation exposure the duplicated genome necessary for recombination), since caffeine-sensitive repair begins immediately and is completed before resumption of DNA synthesis. In contrast, since caffeine-sensitive repair of UV-damage in G1 cells displays a considerable lag and then occurs concomitantly with DNA synthesis, it appears that G1 cells must acquire a second genome in order to accomplish a caffeine-sensitive recovery process. Since a duplicated genome is required for caffeinesensitive repair, all such repair would seem to involve a recombinational mechanism. In G1 cells the process may be a post-replication recombinational mechanism. Since G2 phase cells are considerably more UV-resistant than G1 phase cells, the prereplicative recombinational process appears to be a much more efficient process for dealing with UV-induced damage than the post-replication mechanism.UV-induced mutagenesis was examined in wildtype and rad mutants using a forward mutation system. Rad mutants which show higher UV-induced mutation rates than wild-type retain UV-sensitization by caffeine (and thus presumably retain the recombinational mechanism). In contrast, rad strains which are relatively UV-immutable compared to wild-type do not possess the caffeine-sensitive UV-repair process. The recombinational process therefore may be the major pathway responsible for UV-induced mutation.AECL Reference No. 6251; NRC Publication No. 16999     

Three <Emphasis Type="Italic">nth</Emphasis> homologs are all required for efficient repair of spontaneous DNA damage in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans is a bacterium that can survive extreme DNA damage. To understand the role of endonuclease III (Nth) in oxidative repair and mutagenesis, we constructed nth single, double and triple mutants. The nth mutants showed no significant difference with wild type in both IR resistance and H₂O₂ resistance. We characterized these strains with regard to mutation rates and mutation spectrum using the rpoB/Rif^r system. The Rif^r frequency of mutant MK1 (△dr0289) was twofold higher than that of wild type. The triple mutant of nth (ME3)generated a mutation frequency 34.4-fold, and a mutation rate 13.8-fold higher than the wild type. All strains demonstrated specific mutational hotspots. Each single mutant had higher spontaneous mutation frequency than wild type at base substitution (G:C → A:T). The mutational response was further increased in the double and triple mutants. The higher mutation rate and mutational response in ME3 suggested that the three nth homologs had non-overlapped and overlapped substrate spectrum in endogenous oxidative DNA repair.     

UV-Induced mitotic recombination and its dependence on photoreactivation and liquid holding in the rad6-1 mutant of Saccharomyces cerevisiae

Summary Spontaneous and UV-induced mitotic recombination was compared in diploids homozygous for rad6-1 mutation and in the wild-type strain carrying heterozygous markers for detecting gene conversion (hom2-1, hom2-2) and crossing over (adel, ade2). Diploids homozygous for rad6-1 mutation were characterised by an elevated level of spontaneous and UV-induced mitotic recombination, particularly the intergenic events. Exposure of UV-irradiated strains to visible light resulted in an increased survival and decreased level of mitotic recombination. Liquid holding (LH) differentially affected frequency of mitotic intergenic and intragenic recombination in mutant and wild-type strains, being without any significant effect on cell survival. In a mutant strain intragenic recombination is significantly increased, intergenic only slightly. In the wild-type strain intragenic recombination is slightly decreased but intergenic is not changed by LH. Visible light applied after LH had no effect on survival and mitotic recombination in the wild type, while in the mutant strain photoreactivability of survival was fully preserved and accompanied by a decrease in the frequency of intragenic and intergenic recombination. The results suggest that metabolic pathways responsible for restoring cell survival are independent of or only partly overlapping with those concerning recombination events.     

Genetic control of repair of radiation damage produced under euoxic and anoxic conditions in diploid yeastSaccharomyces cerevisiae

Summary Diploid wild type yeast strains 211, X2180 and the radiation sensitive mutantsrad2, 6, 9, 18, 50–55, and57 were exposed to cobalt-60 gamma radiation, in the presence and absence of oxygen, in order to identify the RAD loci involved in the repair of sublethal damage (SLD), recovery from potentially lethal damage (PLD) and oxygen enhancement ratio (OER). Response of wild type and mutants were compared in terms of survival curve parameters D_q, D₁₀, D₁, and D₀. As compared to wild type the mutants showed increased sensitivity to radiation lethality, both under euoxic and hypoxic conditions, as judged by the reduction in D_q and D₀ values. OER was reduced in therad2, 9, 18, 50, 51, and57 mutants indicating that these genes could be associated with the repair of gamma radiation damage produced under hypoxic condition.Shoulder (D_q) a measure of the ability of the cells to repair SLD, was reduced in therad6, 9, 18, 50, 53, and57 strains and was almost absent in therad51, 52, 54, and55 mutants. The ability to recover from PLD was equal to that of wild type strain in therad2, 6, 9, and18 strains, reduced in therad53, 55, and57 strains and was absent in therad50–52 and54 strains. In the mutants with liquid holding recovery ability, the extent of recovery from PLD produced under euoxic and hypoxic conditions was the same. These observations suggest that different groups of loci are involved in the control of different repair processes and that the expression of therad50–57 loci play a very important role in the repair of ionising radiation damage.On the basis of the liquid holding recovery data presented here and the observations made by others it is suggested that the unrepaired DSB constitute the PLD and that the repair of DSB involves recombination between homologous chromosomes.     

Biochemical and catalytic properties of endo-1,4-β-xylanases from Thermomyces lanuginosus (wild and mutant strains)

Endoxylanases from the thermophilic fungus, Thermomyces lanuginosus ATCC 44008 (cellulase free wild and mutant strains), were purified to homogeneity by anion-exchange and molecular-sieve chromatographic methods. The purified enzymes were monomers with molecular masses of 22 kDa (wild type) and 24 kDa (mutant), estimated by SDS-PAGE and gel filtration. As glycoproteins, the purified enzymes had 0.74% (wild type) and 11.8% (mutant) carbohydrate contents, and pI values of 5.8 and 6, respectively. The optimal pH and temperature values of wild type xylanase were determined to be pH 7 and 60 °C, whereas pH 6.7 and 70 °C, were optimal for the purified mutant enzyme (K _m and V _max values of 3.7 mg ml^–1 and 670 mol min^–1 xylose compared to the kinetic values of the purified wild type xylanase –5.1 mg ml^–1 and 385 mol min^–1 xylose). Inhibition studies suggested the possible involvement of histidine, tryptophan residues and carboxylic groups in the binding or catalysis.     

Characteristics of a photorespiratory mutant of barley (Hordeum vulgare L.) deficient in phosphogly collate phosphatase

A barley mutant RPr84/90 has been isolated by selecting for plants which grow poorly in natural air, but normally in air enriched to 0.8% CO₂. After 5 minutes of photosynthesis in air containing¹⁴CO₂ this mutant incorporated 26% of the¹⁴C carbon into phosphoglycollate, a compound not normally labelled in wild type (cv. Maris Mink) leaves.The activity of phosphoglycollate phosphatase (EC 3.1.1.18) was 1.2 nkat mg^–1 protein at 30°C in RPr 84/90 compared to 19.2 nkat mg^–1 protein in the wild-type leaves. Phosphoglycollate phosphatase activity was not detected after protein separation by electrophoresis of leaf extracts from the mutant on polyacrylamide gels; on linear 5% acrylamide gels three bands with enzyme activity were separated from extracts of wild type plants. Gradient gel electrophoresis followed by activity staining showed two bands in Maris Mink tracks of MW 86,000 and 96,000, but no bands in 84/90. This is the first report of isozymes of phosphoglycollate phosphatase in barley which were absent in the mutant extracts. Our results confirm an earlier report of isozymes of this phosphatase in Phaseolus vulgaris [18].The photosynthetic rate of RPr 84/90 in 1% O₂, 350 l CO₂ l^–1 was 9–12 mg CO₂ dm^–2 h^–1 at 20°C, whereas the wild-type rate was 27–29 mg CO₂ dm^–2 h^–1 at 20°C. In 21% O₂, 350 l CO₂ l^–1 the rate was 2–3 mg CO₂ dm^–2 h^–1 in the mutant and 20 mg CO₂ dm^–2 h^–1 in the wild type.Genetic analysis has shown that the mutation segregates as a single recessive nuclear gene.     

Reactivation of photodynamically inactivated lambda phages

Summary UV-irradiated c phages show a lower survival when plated on rec ^–-cells as compared to rec ⁺-cells. Photodynamically inactivated c phages show the lowest survival on hcr ^–; rec ^–-cells. The rec-functions do not influence the repair of UV- or photodynamically induced lesions in T1 phages.     

Repair of MMS-induced DNA double-strand breaks in haploid cells of Saccharomyces cerevisiae, which requires the presence of a duplicate genome.

Summary The formation and repair of double-strand breaks induced in DNA by MMS was studied in haploid wild type and MMS-sensitive rad6 mutant strains of Saccharomyces cerevisiae with the use of the neutral and alkaline sucrose sedimentation technique. A similar decrease in average molecular weight of double-stranded DNA from 5–6x10⁸ to 1–0.7x10⁸ daltons was observed following treatment with 0.5% MMS in wild type and mutant strains. Incubation of cells after MMS treatment in a fresh drug-free growing medium resulted in repair of double-strand breaks in the wild type strain, but only in the exponential phase of growth. No repair of double-strand breaks was found when cells of the wild type strain were synchronized in G-1 phase by treatment with factor, although DNA single-strand breaks were still efficiently repaired. Mutant rad6 which has a very low ability to repair MMS-induced single-strand breaks, did not repair double-strand breaks regardless of the phase of growth.These results suggest that (1) repair of double-strand breaks requires the ability for single-strand breaks repair, (2) rejoining of double-strand breaks requires the availability of two homologous DNA molecules, this strongly supports the recombinational model of DNA repair.     

Three additional genes involved in pyrimidine dimer removal in Saccharomyces cerevisiae: RAD7, RAD14 and MMS19.

Summary The ability to remove ultraviolet (UV)-induced pyrimidine dimers from the nuclear DNA of yeast was examined in two radiation-sensitive (rad) mutants and one methyl methanesulfonate-sensitive (mms) mutant of the yeast Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by an endonuclease activity prepared from crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad7, rad14 and mms19 mutants were found to be defective in their ability to remove UV-induced dimers from nuclear DNA. All three mutants belong to the same epistatic group as the other mutants involved in excision-repair. All three mutants show enhanced UV-induced mutations. The rad14 mutant also shows epistatic interactions with genes in the other two UV repair pathways.     

Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47–56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O₂ evolving activity without addition of CaCl₂ than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.Abbreviations PS photosystem - chl chlorophyll - LHCP light harvesting chlorophyll a/b protein complex - WT wild type - OEE1, OEE2 and OEE3 oxygen evolution enhancing complex of 29–36 kDa, 21–24 kDa and 16–18 kDa, respectively     

Rice alcohol dehydrogenase 1 promotes survival and has a major impact on carbohydrate metabolism in the embryo and endosperm when seeds are germinated in partially oxygenated water

Background and Aims

Rice (Oryza sativa) has the rare ability to germinate and elongate a coleoptile under oxygen-deficient conditions, which include both hypoxia and anoxia. It has previously been shown that ALCOHOL DEHYDROGENASE 1 (ADH1) is required for cell division and cell elongation in the coleoptile of submerged rice seedlings by means of studies using a rice ADH1-deficient mutant, reduced adh activity (rad). The aim of this study was to understand how low ADH1 in rice affects carbohydrate metabolism in the embryo and endosperm, and lactate and alanine synthesis in the embryo during germination and subsequent coleoptile growth in submerged seedlings.

Methods

Wild-type and rad mutant rice seeds were germinated and grown under complete submergence. At 1, 3, 5 and 7 d after imbibition, the embryo and endosperm were separated and several of their metabolites were measured and compared.

Key results

In the rad embryo, the rate of ethanol fermentation was halved, while lactate and alanine concentrations were 2·4- and 5·7- fold higher in the mutant than in the wild type. Glucose and fructose concentrations in the embryos increased with time in the wild type, but not in the rad mutant. The rad mutant endosperm had lower amounts of the α-amylases RAMY1A and RAMY3D, resulting in less starch degradation and lower glucose concentrations.

Conclusions

These results suggest that ADH1 is essential for sugar metabolism via glycolysis to ethanol fermentation in both the embryo and endosperm. In the endosperm, energy is presumably needed for synthesis of the amylases and for sucrose synthesis in the endosperm, as well as for sugar transport to the embryo.     

UV-sensitivity and repair of UV-damages in Salmonella of wild type.

Summary The UV-sensitivity of wild type Salmonella strains has been compared to that of wild type E. coli and its UV-sensitive mutants. Many wild type Salmonella strains are 4–5 times more sensitive than wild type E. coli and their inactivation curve is similar to that for E. coli with a mutation in the polA gene. Alkaline sucrose gradient centrifugation has shown a deficiency of these strains in normal excision repair of UV-damaged DNA. This deficiency is not a Salmonella genus feature because one strain as resistant as wild type E. coli was found. This resistant strain showed normal excision repair in alkaline sucrose gradient centrifugation experiments. The possible influence of plasmids and mutations in repair genes on the ability of Salmonella to repair UV-damaged DNA is discussed.     

Plasmid control of recombination in E. coli K 12

Summary The recombination proficiency of three recipient strains of Escherichia coli K 12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec ^– derivatives. The same plasmid was also found to protect different rec ^– derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.     

Characterization of long patch excision repair of DNA in ultraviolet-irradiated Escherichia coli: An inducible function under rec-lex control

Summary Excision repair in ultraviolet-irradiated wild-type Escherichia coli produces a bimodal distribution of repair patch sizes in the DNA. Approximately 99% of the repair events result in short patches of 20–30 nucleotides produced by a constitutive repair system. The remaining 1% result in patches which are at least 1,500 nucleotides in length. This long patch repair is shown to be a damage-inducible process under control of the rec-lex regulatory circuit. The kinetics of the two processes differ; short patch synthesis begins immediately after irradiation and is virtually completed prior to synthesis of the majority of the long patches. Long patch repair synthesis is a linear function of UV dose up to a plateau at 60 J/m², and hence each long patch event is the consequence of a single UV-induced lesion. Long patch repair does not appear to be nessarily error-prone, since no alteration in repair synthesis occurs as a result of a mutation umuC ^- which renders cells nonmutable by UV. Evidence is presented suggesting that DNA polymerase I is responsible for both long and short patch synthesis in wild type cells under inducing conditions. In the absence of polymerase I the constitutive patch size averages 80–90 nucleotides, and this distribution is unchanged by induction.     

Pigment protein complexes and functional properties of tetratype resulting from crosses between CP1 and CP2 less Chlamydomonas mutants

A chlorophyll b-less mutant of Chlamydomonas reinhardtii (Pg ₂₇) was isolated after UV irradiation of the wild type cells. This photosynthetically competent mutant totally lacks chlorophyll b and the CP₂ chlorophyll-protein complex. However, SDS-PAGE, proteolytic digestions and immunodetections demonstrated that the 24–25 Kd apoproteins of the lacking CP₂ complex are still present in thylakoids of the Pg₂₇ mutant. It is concluded that this CP₂-less mutant is affected in the biosynthesis pathway of chlorophyll b.This CP₂-less mutant was crossed with a CP₁-less mutant (Fl₅) Fluorescence emission spectra and fluorescence inductions in the presence of DCMU were analysed in the resulting (cp ₂ ^– , cp ₁ ⁺ ), (cp ₂ ⁺ , cp ₁ ^– ), (cp ₂ ⁺ , cp ₁ ⁺ ), cp ₂ ^– , cp ₁ ^– )tetratype. Differences in PS 2 optical cross section and in the relative amplitude or localisation of fluorescence emission peaks fit well with a quadripartite model where PS1 and PS2 would each correspond to a reaction centre core complex (CP₁ and CP₂ respectively) associated to a light harvesting antenna (LHC1 and LHC2 respectively). The occurrence of energy transfers from PS1 peripheral antenna to PS2 in the Fl ₅ mutant shows that, in absence of CP₁, at least a part of its associated PS1 light harvesting antenna migrates in the PS2 containing appressed thylakoids.Abbreviations Chl Chlorophyll - LHC Light harvesting chl a/b complex - CP₂ Predominant form of LHC or SDS polyacrylamide gels - WT Wild type - DM Double mutant (cp ₁ ^– , cp ₂ ^– ) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - DOC-PAGE Deoxycholate polyacrylamide gel electrophoresis     

Effect of seabuckthorn leaf extracts on circulating energy fuels, lipid peroxidation and antioxidant parameters in rats during exposure to cold, hypoxia and restraint (C–H–R) stress and post stress recovery

The present study was carried out to study mechanism of adaptogenic activity of seabuckthorn leaf extract, administered orally in rats both in single and five doses at a dose of 100 mg/kg body weight 30 min prior to C–H–R exposure. The efficacy of the extract was studied on circulating energy fuels, lipid peroxidation and anti-oxidant parameters in rats on attaining the T_rec 23 °C during C–H–R exposure and after recovery (T_rec 37 °C) from C–H–R induced hypothermia. Single dose treatment in rats restricted rise in blood malondialdehyde (MDA) levels and decrease in glutathione (GSH) and catalase (CAT) levels. Both single and five doses also restricted the rise in serum free fatty acids (FFA) and lactate dehydrogenase (LDH) levels on attaining T_rec 23 °C during C–H–R exposure, suggesting more efficient utilization of FFA for energy production and better maintained cell membrane permeability. This suggested that the adaptogenic activity of the extract might be due to its anti-oxidative activity, maintained blood glucose levels, better utilization of FFA and improved cell membrane permeability.     

A Simple and Rapid Quantitative Method of Detection of the Common Achondroplasia Mutation: Analysis in Mismatch Repair Deficient Cells

Achondroplasia is the most common form of dwarfism and has an incidence of approximately 1/7500. In more than 97% of cases, it is caused by a recurrent point mutation, a G to A substitution at nucleotide position 1138 (G1138A) of the fibroblast growth factor receptor 3 gene. Although this is an autosomal dominant condition, more than 90% of all mutations occur sporadically making this one of the most mutagenic sites in the human genome. The reasons for the high spontaneous G1138A mutation rate are not known. This investigation was performed by developing a simple and rapid semi-quantitative allele specific PCR based assay capable of reliably detecting more than 25 mutant G1138A copies in a pool of 300 000 wild type molecules. Using this assay, the G1138A mutation frequency was measured in cell lines deficient in mismatch repair (LoVo, SW48) and comparing it with controls. No differences were found in the frequency of this point mutation between the mismatch repair deficient and wild type cell lines.__________From Genetika, Vol. 41, No. 8, 2005, pp. 1137–1141.Original English Text Copyright © 2005 by Grewal.This article was submitted by the author in English.