Biomedical subjects

Anetta Nowosielska

Publications and source records attributed to Anetta Nowosielska.

6 recordsLinked to original sources

PubMed2004

Spontaneous and cisplatin-induced recombination in Escherichia coli.

To measure cisplatin (cis-diaminodichloroplatinum(II))-induced recombination, we have used a qualitative intrachromosomal assay utilizing duplicate inactive lac operons containing non-overlapping deletions and selection for Lac+ recombinants. The two operons are separated by one Mb and conversion of one of them yields the Lac+ phenotype. Lac+ formation for both spontaneous and cisplatin-induced recombination requires the products of the recA, recBC, ruvA, ruvB, ruvC, priA and polA genes. Inactivation of the recF, recO, recR and recJ genes decreased cisplatin-induced, but not spontaneous, recombination. The dependence on PriA and RecBC suggests that recombination is induced following stalling or collapse of replication forks at DNA lesions to form double strand breaks. The lack of recombination induction by trans-DDP suggests that the recombinogenic lesions for cisplatin are purine-purine intrastrand crosslinks.

Cell Survival↗

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

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↗