Biomedical subjects

Elzbieta Grzesiuk

Publications and source records attributed to Elzbieta Grzesiuk.

11 recordsLinked to original sources

PubMed2006

Mutator specificity of Escherichia coli alkB117 allele.

The Escherichia coli AlkB protein encoded by alkB gene was recently found to repair cytotoxic DNA lesions 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) by using a novel iron-catalysed oxidative demethylation mechanism that protects the cell from the toxic effects of methylating agents. Mutation in alkB results in increased sensitivity to MMS and elevated level of MMS-induced mutations. The aim of this study was to analyse the mutational specificity of alkB117 in a system developed by J.H. Miller involving two sets of E. coli lacZ mutants, CC101-106 allowing the identification of base pair substitutions, and CC107-CC111 indicating frameshift mutations. Of the six possible base substitutions, the presence of alkB117 allele led to an increased level of GC-->AT transitions and GC-->TA and AT-->TA transversions. After MMS treatment the level of GC-->AT transitions increased the most, 22-fold. Among frameshift mutations, the most numerous were -2CG, -1G, and -1A deletions and +1G insertion. MMS treatment appreciably increased all of the above types of frameshifts, with additional appearance of the +1A insertion.

Alleles↗

PubMed2006

Gut myoelectrical activity induces heat shock response in Escherichia coli and Caco-2 cells.

The heat shock response is associated with the intracellular expression of a number of highly conserved heat shock proteins (Hsps). According to their molecular size, Hsps have been divided into several groups, which are strongly conserved and show high homology between the species, e.g., Hsp70, MW 70 kDa (Lindquist & Craig, 1998; Morimoto, 1998; Jolly & Morimoto, 2000; Zylicz et al. 2001). In all organisms the Hsp expression under stress conditions is regulated at transcriptional level, e.g., in humans by the heat shock transcription factor Hsf1 (Morimoto, 1998; Wu, 1995), while in Escherichia coli by replacement of the sigma factor sigma(70) in RNA polymerase by the sigma factor sigma(32) (Gross, 1987). The Hsps allow cell survival under stress conditions by renaturating of denaturated proteins, protecting of stress-labile proteins, preventing protein aggregation (chaperone functions), and by degradation of damaged proteins (protease activities) (Lindquist & Craig, 1988; Morimoto, 1998; Jolly & Morimoto, 2000). They have also many housekeeping functions under non-stressful conditions during the cell cycle, growth, development, and differentiation (Morimoto, 1998). Among a number of plausible inducing factors already studied, extremely low artificial electromagnetic fields have been shown to induce stress response in various cells, such as expression of sigma(32) mRNA (Cairo et al. 1998) and induction of DnaJ and DnaK proteins in Eschericha coli (Chow & Tung, 2000); expression of hsp-16 gene in Caenorhabditis elegans (Miyakawa et al., 2001); induction of heat shock transcription factor Hsf1 and Hsp70, Hsp90 and Hsp27 in human cells (Lin et al. 1997; Lin et al. 1998; Goodman & Blank, 1998; Pipkin et al. 1999). Nevertheless, the role of endogenous electromagnetic fields, i.e., generated by electrically active cells within a body remains controversial. Heat shock proteins (Hsps) protect cells against various environmental and endogenous stressors. Cytoprotection caused by Hsps involves tolerance induced by one agent against other, more severe agents. We have found that exposure of prokaryotic (Escherichia coli) and eukaryotic (Caco-2) cells to an electrical field (EF) connected with a myoelectrical migrating complex (MMC) generated by the small intestine smooth muscle induces the heat shock response. Using Western blot analysis, we have detected an elevated level of sigma factor 32 in E. coli cells exposed to MMC-related EF, and confocal microscopy indicated an increased level of the inducible form of Hsp70 protein in EF-stimulated Caco-2 cells. Additionally, we have found that this induced level of Hsp70 protected the Caco-2 cells against apoptosis caused by camptothecin. Our observations suggest that the myoelectrical activity of the gut may induce heat shock mechanisms in the cells of gut epithelium as well as in gastrointestinal micro-organisms.

Action Potentials↗

PubMed2005

AlkB dioxygenase in preventing MMS-induced mutagenesis in Escherichia coli: effect of Pol V and AlkA proteins.

The deleterious effect of defective alkB allele encoding 1meA/3meC dioxygenase on reactivation of MMS-treated phage DNA has been frequently studied. Here, it is shown that: (i) AlkB protects the cells not only against the genotoxic but also against the potent mutagenic activity of MMS; (ii) mutations arising in alkB-defected strains are umuDC-dependent, and deletion of umuDC dramatically reduce MMS-induced mutations resulting from the presence of 1meA/3meC in DNA; (iii) specificity of MMS-induced argE3-->Arg+ reversions in AB1157 alkB-defective cells are predominantly AT-->TA transversions and GC-->AT transitions; (iv) overproduction of AlkA and the resultant decrease in 3meA residues in DNA dramatically reduce MMS-induced mutations. This reduction is most probably a secondary effect of AlkA due to a decrease in 3meA residues in DNA and, in consequence, suppression of SOS induction and Pol V expression. Overproduction of UmuD'C proteins reverses this effect.

Arginine↗

PubMed2005

Mutator activity and specificity of Escherichia coli dnaQ49 allele--effect of umuDC products.

The high fidelity of DNA replication in Escherichia coli is ensured by the alpha (DnaE) and epsilon (DnaQ) subunits of DNA polymerase providing insertion fidelity, 3'-->5' exonuclease proofreading activity, and by the dam-directed mismatch repair system. dnaQ49 is a recessive allele that confers a temperature-sensitive proofreading phenotype resulting in a high rate of spontaneous mutations and chronic induction of the SOS response. The aim of this study was to analyse the mutational specificity of dnaQ49 in umuDC and DeltaumuDC backgrounds at 28 and 37 degrees C in a system developed by J.H. Miller. We confirmed that the mutator activity of dnaQ49 was negligible at 28 degrees C and fully expressed at 37 degrees C. Of the six possible base pair substitutions, only GC-->AT transitions and GC-->TA and AT-->TA transversions were appreciably increased. However, the most numerous mutations were frameshifts, -1G deletions and +1A insertions. All mutations which increased in response to dnaQ49 damage were to a various extent umuDC-dependent, especially -1G deletions. This type of mutations decreased in CC108dnaQ49DeltaumuDC to 10% of the value found in CC108dnaQ49umuDC+ and increased in the presence of plasmids producing UmuD'C or UmuDC proteins. In the recovery of dnaQ49 mutator activity the plasmid harbouring umuD'C genes was more effective than the one harbouring umuDC. Analysis of mutational specificity of pol III with defective epsilon subunit indicates that continuation of DNA replication is allowed past G:T, C:T, T:T (or C:A, G:A, A:A) mismatches but does not allow for acceptance of T:C, C:C, A:C (or A:G, G:G, T:G) (the underlined base is in the template strand).

Alleles↗

PubMed2005

Effect of SOS-induced Pol II, Pol IV, and Pol V DNA polymerases on UV-induced mutagenesis and MFD repair in Escherichia coli cells.

Irradiation of organisms with UV light produces genotoxic and mutagenic lesions in DNA. Replication through these lesions (translesion DNA synthesis, TSL) in Escherichia coli requires polymerase V (Pol V) and polymerase III (Pol III) holoenzyme. However, some evidence indicates that in the absence of Pol V, and with Pol III inactivated in its proofreading activity by the mutD5 mutation, efficient TSL takes place. The aim of this work was to estimate the involvement of SOS-inducible DNA polymerases, Pol II, Pol IV and Pol V, in UV mutagenesis and in mutation frequency decline (MFD), a mechanism of repair of UV-induced damage to DNA under conditions of arrested protein synthesis. Using the argE3-->Arg(+) reversion to prototrophy system in E. coli AB1157, we found that the umuDC-encoded Pol V is the only SOS-inducible polymerase required for UV mutagenesis, since in its absence the level of Arg(+) revertants is extremely low and independent of Pol II and/or Pol IV. The low level of UV-induced Arg(+) revertants observed in the AB1157mutD5DumuDC strain indicates that under conditions of disturbed proofreading activity of Pol III and lack of Pol V, UV-induced lesions are bypassed without inducing mutations. The presented results also indicate that Pol V may provide substrates for MFD repair; moreover, we suggest that only those DNA lesions which result from umuDC-directed UV mutagenesis are subject to MFD repair.

DNA Repair↗

PubMed2004

Effect of deletion of SOS-induced polymerases, pol II, IV, and V, on spontaneous mutagenesis in Escherichia coli mutD5.

The E. coli dnaQ gene encodes the epsilon subunit of DNA polymerase III (pol III) responsible for the proofreading activity of this polymerase. The mutD5 mutant of dnaQ chronically expresses the SOS response and exhibits a mutator phenotype. In this study we have constructed a set of E. coli AB1157 mutD5 derivatives deleted in genes encoding SOS-induced DNA polymerases, pol II, pol IV, and pol V, and estimated the frequency and specificity of spontaneous argE3-->Arg(+) reversion in exponentially growing and stationary-phase cells of these strains. We found that pol II exerts a profound effect on the specificity of spontaneous mutation in exponentially growing cells. Analysis of growth-dependent Arg(+) revertants in mutD5 polB(+) strains revealed that Arg(+) revertants were due to tRNA suppressor formation, whereas those in mutD5 DeltapolB strains arose by back mutation at the argE3 ochre site. In stationary-phase bacteria, Arg(+)revertants arose mainly by back mutation, regardless of whether they were proficient or deficient in pol II. Our results also indicate that in a mutD5 background, the absence of pol II led to increased frequency of Arg(+) growth-dependent revertants, whereas the lack of pol V caused its dramatic decrease, especially in mutD5 DeltaumuDC and mutD5 DeltaumuDC DeltapolB strains. In contrast, the rate of stationary-phase Arg(+)revertants increased in the absence of pol IV in the mutD5 DeltadinB strain. We postulate that the proofreading activity of pol II excises DNA lesions in exponentially growing cells, whereas pol V and pol IV are more active in stationary-phase cultures.

DNA Polymerase III↗

PubMed2004

Spontaneous mutagenesis in exponentially growing and stationary-phase, umuDC-proficient and -deficient, Escherichia coli dnaQ49.

Spontaneous mutations arise not only in exponentially growing bacteria but also in non-dividing or slowly dividing stationary-phase cells. In the latter case mutations are called adaptive or stationary-phase mutations. High spontaneous mutability has been observed in temperature sensitive Escherichia coli dnaQ49 strain deficient in 3'-->5' proofreading activity assured by the e subunit of the main replicative polymerase, Pol III. The aim of this study was to evaluate the effects of the dnaQ49 mutation and deletion of the umuDC operon encoding polymerase V (Pol V) on spontaneous mutagenesis in growing and stationary-phase E. coli cells. Using the argE3(OC) -->Arg+ reversion system in the AB1157 strain, we found that the level of growth-dependent and stationary-phase Arg+ revertants was significantly increased in the dnaQ49 mutant at the non-permissive temperature of 37 degrees C. At this temperature, in contrast to cultures grown at 28 degrees C, SOS functions were dramatically increased. Deletion of the umuDC operon in the dnaQ49 strain led to a 10-fold decrease in the level of Arg+ revertants in cultures grown at 37 degrees C and only to a 2-fold decrease in cultures grown at 28 degrees C. Furthermore, in stationary-phase cultures Pol V influenced spontaneous mutagenesis to a much lesser extent than in growing cultures. Our results indicate that the level of Pol III desintegration, dependent on the temperature of incubation, is more critical for spontaneous mutagenesis in stationary-phase dnaQ49 cells than the presence or absence of Pol V.

DNA Polymerase III↗

PubMed2003

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.

Bacterial Proteins↗

PubMed2003

E. coli BW535, a triple mutant for the DNA repair genes xth, nth, and nfo, chronically induces the SOS response.

A strong chronic induction of the SOS response system occurs in E. coli BW535, a strain defective in nth, nfo and xth genes, and hence severely deficient in the repair of abasic sites in DNA. This was shown here by visualization of filamentous growth of the BW535 strain and by measuring the level of beta-galactosidase expressed in BW535/pSK1002 in comparison to the AB1157/pSK1002 strain. The plasmid pSK1002 bears an umuC::lacZ fusion in which lacZ is under the control of the umuC promoter and regulated under the SOS regulon. Increases in the expression of beta-galactosidase occur in BW535 without any exogenous SOS inducer. Chronic induction of the SOS response was observed previously in E. coli strains bearing mutations in certain genes that have mutator activity and BW535 is a moderate mutator strain. However, not all mutators show this property, since chronic induction of SOS was not observed in mutT or mutY mutators. MutT and MutY proteins, when active, protect bacteria from mutations induced by 8-oxoG lesions in DNA. This suggests that accumulation of abasic sites, but not 8-oxoG residues in DNA, induce the SOS response.

Carbon-Oxygen Lyases↗

PubMed2003

[Adaptive mutations].

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Adaptation, Physiological↗

PubMed2002

Induction of the SOS response in starved Escherichia coli.

The SOS system in Escherichia coli is induced in response to DNA damage and the arrest of DNA synthesis. Here we show that in AB1157 bacteria starved for arginine, conditions for induction of adaptive mutations, the LexA-dependent SOS system is induced, but that this occurs only when the bacteria resume growth and when the source of carbon is glycerol rather than glucose (glycerol, but not glucose, enables synthesis of cAMP). Therefore, we conclude that starved cells accumulate some lesions in DNA, which in growth conditions may trigger SOS induction by a process that is cAMP-dependent.

Actin Cytoskeleton↗