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DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair
Get PDFHigh accuracy (fidelity) of DNA replication is important for cells to preserve the genetic identity and to prevent the accumulation of deleterious mutations. The error rate during DNA replication is as low as 10�9 to 10�11 errors per
base pair. How this low level is achieved is an issue of major interest. This review is concerned with the mechanisms underlying the fidelity of the chromosomal replication in the model system Escherichia coli by DNA polymerase III holoenzyme, with further emphasis on participation of the other, accessory DNA polymerases, of which E. coli contains four (Pols I, II, IV, and V).
Detailed genetic analysis of mutation rates revealed that (1) Pol II has an important role as a back-up proofreader for Pol III, (2) Pols IV and V do not normally contribute significantly to replication fidelity, but can readily do so
under conditions of elevated expression, (3) participation of Pols IV and V, in contrast to that of Pol II, is specific to the lagging strand, and (4) Pol I also
makes a lagging-strand-specific fidelity contribution, limited, however, to the faithful filling of the Okazaki fragment gaps. The fidelity role of the Pol III �subunit is also reviewed
Effects of Roscovitine on Schedule of Divisional Morphogenesis, Basal Bodies Proliferation and Cell Divisions in Tetrahymena thermophila
Get PDFSummary. During cell cycle of a ciliate Tetrahymena thermophila the divisions of micro- and macronucleus, cortical morphogenesis and '
cytokinesis are temporarily coordinated. Cortical morphogenesis begins with proliferation of the new ciliary basal bodies (BBs) within
meridional cortical rows of ciliary BBs, and with the local proliferation of BBs, which form the new oral apparatus (OA2), positioned subequatorialy
and destined for prospective posterior daughter cell (opisthe). Prior to cytokinesis, two prospective daughter cells are of equal
size and show metamery of their cortical patterns. We studied effects of 20 uM roscovitine (an inhibitor of several cyclin-dependent kinases)
on the cell cycle progression of T. thermophila. We showed that roscovitine delayed cell division, delayed or arrested macronuclear division
and induced increase of cell size and the number of BBs in the cortical rows. The increase in the number of BBs in cortical rows induced
cell elongation which was proportional to the increase in cell surface area. There was uncoupling between this BBs_proliferation which
is continued during prolonged cell cycle and delayed cytokinesis, what resulted in topological alteration of the respective positions of the
OA2 and of the contractile vacuole pores (CVPs). In roscovitine treated cells, the new OA2 was positioned subequatorialy, but the fission
zone was shifted posterior to the equatorial plane of the cell and positioned across and in the extreme cases behind of the new OA2. This
resulted in the formation of a large proter and small size opisthe. The roscovitine treatment induced a formation of a plethora of phenotypes
of postdividing cells. We found that irrespective of changes in divisional morphogenesis induced by roscovitine treatment, all mature BBs
were associated with the cdc!4-like phosphatase. Taken together all these data indicate that during cell cycle of T. thermophila the normal
morphology of the daughter cells depends on the proper division of micro- and macronucleus and on temporal control of BBs proliferation
along the longitudinal rows, during OA2 stomatogenesis and during selection of BBs involved in differentiation of apical BBs (couplets)
and cell division.
Key words: Tetrahymena, roscovitine, basal bodies, morphogenesis, macronucleus, cdc!4p.
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Chronic myeloid leukemia stem cells display alterations in expression of genes involved in oxidative phosphorylation.
Get PDFAbstract The mitochondrial respiratory chain (MRC) consists of protein complexes I, II, III, IV and V that support oxidative phosphorylation (OXPHOS), which depends on electron transport to generate adenosine triphosphate (ATP). Electron "leakage" from the MRC generates reactive oxygen species (ROS). Chronic myeloid leukemia in chronic phase (CML-CP) stem cells (LSCs) produce high levels of mitochondrial ROS, causing oxidative DNA damage, resulting in genomic instability, generating imatinib-resistant BCR-ABL1 kinase mutants and additional chromosomal aberrations. Using global mRNA microarray analysis combined with Ingenuity Pathway Analysis we found that LSCs display enhanced expression of genes encoding MRC complexes I, II, IV and V. However, expression of genes encoding complex III was deregulated. Treatment with imatinib did not correct the aberrant levels of MRC genes. Therefore we postulate that abnormal expression of MRC genes may facilitate electron "leakage" to promote the production of ROS and accumulation of genomic instability in LSCs in imatinib-naive and imatinib-treated patients
Defining the pathogenesis of human mtDNA mutations using a yeast model : the case of T8851C
Get PDFMore and more mutations are found in the mitochondrial DNA of various patients but ascertaining their pathogenesis is often difficult. Due to the conservation of mitochondrial function from yeast to humans, the unique ability of yeast to survive without production of ATP by oxidative phosphorylation, and the amenability of the yeast mitochondrial genome to site-directed mutagenesis, yeast is an excellent model for investigating the consequences of specific human mtDNA mutations. Here we report the construction of a yeast model of a point mutation (T8851C) in the mitochondrially-encoded subunit a/6 of the ATP synthase that has been associated with bilateral striatal lesions, a group of rare human neurological disorders characterized by symmetric degeneration of the corpus striatum. The biochemical consequences of this mutation are unknown. The T8851C yeast displayed a very slow growth phenotype on non-fermentable carbon sources, both at 28°C (the optimal temperature for yeast growth) and at 36°C. Mitochondria from T8851C yeast grown in galactose at 28°C showed a 60% deficit in ATP production. When grown at 36°C the rate of ATP synthesis was below 5% that of the wild-type, indicating that heat renders the mutation much more deleterious. At both growth temperatures, the mutant F1FO complex was correctly assembled but had only very weak ATPase activity (about 10% that of the control), both in mitochondria and after purification. These findings indicate that a block in the proton-translocating domain of the ATP synthase is the primary cause of the neurological disorder in the patients carrying the T8851C mutation
Nitric oxide and glutathione impact the expression of iron uptake- and iron transport-related genes as well as the content of metals in A. thaliana plants grown under iron deficiency
Get PDFMounting evidence indicate that nitric oxide (NO) acts as a signaling molecule mediating iron deficiency responses through the up-regulation of the expression of iron uptake-related genes. Accordingly, NO donors such as nitrosoglutathione (GSNO) were reported to improve the
fitness of plants grown under iron deficiency. Here, we showed that glutathione, a by-product of GSNO, triggered the up-regulation of the expression of iron uptake- and transport-related gene and an increase of iron concentration in Arabidopsis thaliana seedlings facing iron
deficiency. Furthermore, we provided evidence that under iron deficiency, NO released by GSNO did not improve the root iron concentration but impacted the content of copper.
Collectively, our data highlight the complexity of interpreting data based on the use of NO donors when investigating the role of NO in iron homeostasis
The generation of oxidative stress-induced rearrangements in Saccharomyces cerevisiae mtDNA is dependent on the Nuc1 (EndoG/ExoG) nuclease and is enhanced by inactivation of the MRX complex.
Get PDFOxidative stress is known to enhance the frequency of two major types of alterations in the mitochondrial genome of Saccharomyces cerevisiae: point mutations and large deletions resulting in the generation of respiration-deficient petite rhō mutants. We investigated the effect of antimycin A, a well-known agent inducing oxidative stress, on the stability of mtDNA. We show that antimycin enhances exclusively the generation of respiration-deficient petite mutants and this is accompanied by a significant increase in the level of reactive oxygen species (ROS) and in a marked drop of cellular ATP. Whole mitochondrial genome sequencing revealed that mtDNAs of antimycin-induced petite mutants are deleted for most of the wild-type sequence and usually contain one of the active origins of mtDNA replication: ori1, ori2 ori3 or ori5. We show that the frequency of antimycin-induced rhō mutants is significantly elevated in mutants deleted either for the RAD50 or XRS2 gene, both encoding the components of the MRX complex, which is known to be involved in the repair of double strand breaks (DSBs) in DNA. Furthermore, enhanced frequency of rhō mutants in cultures of antimycin-treated cells lacking Rad50 was further increased by the simultaneous absence of the Ogg1 glycosylase, an important enzyme functioning in mtBER. We demonstrate also that rad50Δ and xrs2Δ deletion mutants display a considerable reduction in the frequency of allelic mitochondrial recombination, suggesting that it is the deficiency in homologous recombination which is responsible for enhanced rearrangements of mtDNA in antimycin-treated cells of these mutants. Finally, we show that the generation of large-scale mtDNA deletions induced by antimycin is markedly decreased in a nuc1Δ mutant lacking the activity of the Nuc1 nuclease, an ortholog of the mammalian mitochondrial nucleases EndoG and ExoG. This result indicates that the nuclease plays an important role in processing of oxidative stress-induced lesions in the mitochondrial genome
Synergistic neurotoxicity of oxygen-glucose deprivation and tetrabromobisphenol A in vitro: role of oxidative stress.
Get PDFThese results suggest the involvement of oxidative stress in the synergistic neurotoxic effects of TBBPA and OGD
Genome Sequence of the Probiotic Strain Lactobacillus rhamnosus (Formerly Lactobacillus casei) LOCK900.
Get PDFLactobacillus rhamnosus LOCK900 fulfills the criteria required for probiotic strains. In this study, we report a whole-genome sequence of this isolate and compare it with other L. rhamnosus complete genome sequences already published
Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal
Get PDFOne of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms
cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to
G>C>>A>T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we
tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was
treated in vitro with HNE, and which was grown in uvrA- E. coli strains, carrying one, two or
all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial
host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower
than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were
present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II.
Mutation spectrum established for strains expressing only Pol V, showed that in uvrA- bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T → C:G, A:T → G:C, G:C → A:T and G:C → T:A prevailed.
The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent
Cascade cell lyses and DNA extraction for identification of genes and microorganisms in kefir grains
Get PDFKefir is a dairy product popular in many countries in Central Europe, especially in Poland and other
countries of Eastern and Northern Europe. This type of fermented milk is produced by a complex
population of symbiotic bacteria and yeasts. In this work, conditions for DNA extraction, involving
disruption of kefir grains and a cascade of cell lysis treatments, were established. Extraction procedure
of total microbial DNA was carried out directly from fresh kefir grains. Using different lysis stringency
conditions, five DNA pools were obtained. Genetic diversity of DNA pools were validated by RAPD
analysis, which showed differences in patterns of amplified DNA fragments, indicating diverse
microbial composition of all the analysed samples. These DNA pools were used for construction of
genomic DNA libraries for sequencing. As much as 50% of the analysed nucleotide sequences
showed homology to sequences from bacteria belonging to the Lactobacillus genus. Several
sequences were similar to sequences from bacteria representing Lactococcus, Oenococcus,
Pediococcus, Streptococcus and Leuconostoc species. Among homologues of yeast proteins were
those fromCandida albicans, Candida glabrata, Kluyveromyces lactis and Saccharomyces cerevisiae.
In addition, several sequences were found to be homologous to sequences from bacteriophages