scholarly journals DNA SEQUENCE ANALYSIS OF MUTAGENICITY AND SITE SPECIFICITY OF ETHYL METHANESULFONATE IN Uvr+ AND UvrB- STRAINS OF ESCHERICHIA COLI

Genetics ◽  
1986 ◽  
Vol 113 (4) ◽  
pp. 811-819
Author(s):  
Philip A Burns ◽  
Frances L Allen ◽  
Barry W Glickman
Keyword(s):  
Dna Sequence ◽  
Excision Repair ◽  
Base Sequence ◽  
Site Specificity ◽  
Repair System ◽  
Induced Mutations ◽  
Base Pairs ◽  
E Coli ◽  
Laci Gene ◽  
Induced Mutagenesis

ABSTRACT EMS-induced mutations within a 180 base pair region of the lacI gene of E. coli were cloned and sequenced. In total, 105 and 79 EMS-induced mutations from a Uvr+ and a UvrB- strain, respectively, were sequenced. The specificity of EMS-induced mutagenesis was very similar in the two strians; G:C → A:T transitions accounted for all but three of the mutants. The overall frequency of induced mutation was fivefold higher in the UvrB- strain compared to the Uvr+ strain. This demonstrates, at the DNA sequence level, that the presumed pre-mutagenic lesion, O  6-ethylguanine, is subject to repair by the uvrABC excision repair system of E. coli. An analysis of mutation frequencies with respect to neighboring base sequence, in the two strains, shows that O  6-ethylguanine lesions adjacent to A:T base pairs present better targets for the excision repair machinery than those not adjacent to A:T base pairs.

Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 661-668
Author(s):  
Mandy Kim ◽  
Erika Wolff ◽  
Tiffany Huang ◽  
Lilit Garibyan ◽  
Ashlee M Earl ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Deinococcus Radiodurans ◽  
Genetic System ◽  
Base Change ◽  
Base Substitution ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
Radiation Induced ◽  
Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

scholarly journals Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

2021 ◽  
Vol 12 ◽  
Author(s):  
Almaz Nigatu Tesfahun ◽  
Marina Alexeeva ◽  
Miglė Tomkuvienė ◽  
Aysha Arshad ◽  
Prashanna Guragain ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Excision Repair ◽  
Dna Lesions ◽  
Base Pairs ◽  
E Coli ◽  
Thymine Glycol ◽  
Base Excision ◽  
The Cost ◽  
Primary Substrate

DNA polymerase III mis-insertion may, where not corrected by its 3′→ 5′ exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The kcat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The KM values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

scholarly journals UV- and MMS-induced mutagenesis of lambdaO(am)8 phage under nonpermissive conditions for phage DNA replication.

2003 ◽  
Vol 50 (4) ◽  
pp. 921-939 ◽  
Author(s):  
Joanna Krwawicz ◽  
Anna Czajkowska ◽  
Magdalena Felczak ◽  
Irena Pietrzykowska
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Protein S ◽  
Dna Lesions ◽  
E Coli ◽  
Phage Dna ◽  
Induced Mutagenesis ◽  
C Proteins ◽  
Inducible Protein

Mutagenesis in Escherichia coli, a subject of many years of study is considered to be related to DNA replication. DNA lesions nonrepaired by the error-free nucleotide excision repair (NER), base excision repair (BER) and recombination repair (RR), stop replication at the fork. Reinitiation needs translesion synthesis (TLS) by DNA polymerase V (UmuC), which in the presence of accessory proteins, UmuD', RecA and ssDNA-binding protein (SSB), has an ability to bypass the lesion with high mutagenicity. This enables reinitiation and extension of DNA replication by DNA polymerase III (Pol III). We studied UV- and MMS-induced mutagenesis of lambdaO(am)8 phage in E. coli 594 sup+ host, unable to replicate the phage DNA, as a possible model for mutagenesis induced in nondividing cells (e.g. somatic cells). We show that in E. coli 594 sup+ cells UV- and MMS-induced mutagenesis of lambdaO(am)8 phage may occur. This mutagenic process requires both the UmuD' and C proteins, albeit a high level of UmuD' and low level of UmuC seem to be necessary and sufficient. We compared UV-induced mutagenesis of lambdaO(am)8 in nonpermissive (594 sup+) and permissive (C600 supE) conditions for phage DNA replication. It appeared that while the mutagenesis of lambdaO(am)8 in 594 sup+ requires the UmuD' and C proteins, which can not be replaced by other SOS-inducible protein(s), in C600 supE their functions may be replaced by other inducible protein(s), possibly DNA polymerase IV (DinB). Mutations induced under nonpermissive conditions for phage DNA replication are resistant to mismatch repair (MMR), while among those induced under permissive conditions, only about 40% are resistant.

scholarly journals Defects in the Error Prevention Oxidized Guanine System Potentiate Stationary-Phase Mutagenesis in Bacillus subtilis

2008 ◽  
Vol 191 (2) ◽  
pp. 506-513 ◽  
Author(s):  
Luz E. Vidales ◽  
Lluvia C. Cárdenas ◽  
Eduardo Robleto ◽  
Ronald E. Yasbin ◽  
Mario Pedraza-Reyes
Keyword(s):  
Oxidative Stress ◽  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Repair System ◽  
Induced Mutations ◽  
General Role ◽  
Error Prevention ◽  
Repair Mechanisms ◽  
Induced Mutagenesis ◽  
Oxidized Guanine

ABSTRACT Previous studies showed that a Bacillus subtilis strain deficient in mismatch repair (MMR; encoded by the mutSL operon) promoted the production of stationary-phase-induced mutations. However, overexpression of the mutSL operon did not completely suppress this process, suggesting that additional DNA repair mechanisms are involved in the generation of stationary-phase-associated mutants in this bacterium. In agreement with this hypothesis, the results presented in this work revealed that starved B. subtilis cells lacking a functional error prevention GO (8-oxo-G) system (composed of YtkD, MutM, and YfhQ) had a dramatic propensity to increase the number of stationary-phase-induced revertants. These results strongly suggest that the occurrence of mutations is exacerbated by reactive oxygen species in nondividing cells of B. subtilis having an inactive GO system. Interestingly, overexpression of the MMR system significantly diminished the accumulation of mutations in cells deficient in the GO repair system during stationary phase. These results suggest that the MMR system plays a general role in correcting base mispairing induced by oxidative stress during stationary phase. Thus, the absence or depression of both the MMR and GO systems contributes to the production of stationary-phase mutants in B. subtilis. In conclusion, our results support the idea that oxidative stress is a mechanism that generates genetic diversity in starved cells of B. subtilis, promoting stationary-phase-induced mutagenesis in this soil microorganism.

N6-Methoxyadenine-Pyrimidine Base Pairs as Substrates for the Mismatch Repair System of Escherichia coli

2001 ◽  
Vol 66 (7) ◽  
pp. 1107-1124 ◽  
Author(s):  
Josef Jiricny
Keyword(s):  
Repair System ◽  
Base Pairs ◽  
Thermal Stabilities ◽  
E Coli ◽  
Template Strand ◽  
H Nmr ◽  
Tautomeric Forms ◽  
Mismatch Repair System

The availability of nucleoside analogues with ambiguous base-pairing properties would be of considerable value in molecular biology. We have incorporated deoxynebularine [9-(2-deoxy-β-D-ribofuranosyl)purine, P], deoxyinosine [9-(2-deoxy-β-D-ribofuranosyl)-6-hydroxypurine, I) and [9-(2-deoxy-β-D-ribofuranosyl)-6-methoxyaminopurine, MeOA] into hexadecamer oligodeoxyribonucleotides and tested their behaviour in DNA•DNA hybridisations in vitro, as well as in oligonucleotide-directed mutagenesis experiments in vivo. The results showed that P behaved as an adenine analogue in all assays. Oligonucleotide duplexes containing I/C or I/T base pairs displayed similar thermal stabilities in DNA•DNA hybridisation experiments, however, during DNA synthesis in vitro and in vivo, hypoxanthine behaved strictly as a guanine analogue. Only MeOA was truly ambiguous in all assays. The 1H NMR spectrum of the nucleoside demonstrated the existence of two distinct tautomeric forms in a ratio of ca 8 : 2, implying that the base might pair with both C and T. Indeed, within the context of synthetic hexadecamer duplexes, MeOA/C and MeOA/T pairs brought about a similar thermal destabilisation, with the former base pair being only marginally less favoured. When used as hybridisation probes on single-stranded M13 DNA, the MeOA-containing hexadecamer oligonucleotides were shown to bind with similar efficiencies to target sequences containing either C or T opposite the analogue. Interestingly, when MeOA is in the template strand during DNA replication, the polymerase III holoenzyme of E. coli reads it predominantly as a G, which indicates that MeOA exists in B-DNA mostly as the anti-imino tautomer.

scholarly journals Excision repair functions in Saccharomyces cerevisiae recognize and repair methylation of adenine by the Escherichia coli dam gene.

1986 ◽  
Vol 6 (10) ◽  
pp. 3555-3558 ◽  
Author(s):  
M F Hoekstra ◽  
R E Malone
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Excision Repair ◽  
Yeast Cells ◽  
Pyrimidine Dimer ◽  
Repair System ◽  
E Coli

Unlike the DNA of higher eucaryotes, the DNA of Saccharomyces cerevisiae (bakers' yeast) is not methylated. Introduction of the Escherichia coli dam gene into yeast cells results in methylation of the N-6 position of adenine. The UV excision repair system of yeast cells specifically responds to the methylation, suggesting that it is capable of recognizing modifications which do not lead to major helix distortion. The UV repair functions examined in this report are involved in the incision step of pyrimidine dimer repair. These observations may have relevance to the rearrangements and recombination events observed when yeast or higher eucaryotic cells are transformed or transfected with DNA grown in E. coli.

Excision repair functions in Saccharomyces cerevisiae recognize and repair methylation of adenine by the Escherichia coli dam gene

1986 ◽  
Vol 6 (10) ◽  
pp. 3555-3558
Author(s):  
M F Hoekstra ◽  
R E Malone
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Excision Repair ◽  
Yeast Cells ◽  
Pyrimidine Dimer ◽  
Repair System ◽  
E Coli

Unlike the DNA of higher eucaryotes, the DNA of Saccharomyces cerevisiae (bakers' yeast) is not methylated. Introduction of the Escherichia coli dam gene into yeast cells results in methylation of the N-6 position of adenine. The UV excision repair system of yeast cells specifically responds to the methylation, suggesting that it is capable of recognizing modifications which do not lead to major helix distortion. The UV repair functions examined in this report are involved in the incision step of pyrimidine dimer repair. These observations may have relevance to the rearrangements and recombination events observed when yeast or higher eucaryotic cells are transformed or transfected with DNA grown in E. coli.

scholarly journals Suppressible and nonsuppressible +1 G-C base pair insertions induced by ICR-170 at the his4 locus in Saccharomyces cerevisiae.

1985 ◽  
Vol 5 (9) ◽  
pp. 2247-2256 ◽  
Author(s):  
L Mathison ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Base Pair ◽  
Sequence Data ◽  
Induced Mutations ◽  
Coding Region ◽  
Base Pairs ◽  
Sequence Recognition ◽  
Frameshift Suppression ◽  
Target Sites

Fifteen independent ICR-170-induced his4 mutations in Saccharomyces cerevisiae were examined by DNA sequence analysis. All of the mutations contained a +1 G-C base pair addition in the HIS4 coding region. Eleven different sites of insertion were identified. Combined with previous DNA sequence data, 21 ICR-170-induced his4 mutations distributed at 16 different sites were analyzed. The insertions were always located in a consecutive run of two or more G-C base pairs, with all base pairs in each run having identical orientation. Long consecutive G-C runs were preferred target sites over short runs. Although some consecutive G-C runs appeared to be preferred target sites over others of identical length, such preference was not due to any particular type of nucleotide pair immediately adjacent to a given target site. In addition, DNA sequence analyses of the his4 mutations provided a basis for examining the mechanism of mRNA sequence recognition by extragenic suppressors of ICR-170-induced mutations. The implications of these results for mechanisms of frameshift suppression are discussed.

scholarly journals The antimutagenic effect of monoterpenes against UV-irradiation-, 4NQO- and t-BOOH-induced mutagenesis in coli

2011 ◽  
Vol 63 (1) ◽  
pp. 117-128 ◽  
Author(s):  
Biljana Nikolic ◽  
Dragana Mitic-Culafic ◽  
Branka Vukovic-Gacic ◽  
Jelena Knezevic-Vukcevic
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Uv Irradiation ◽  
Mutagenic Effect ◽  
Evolutionary Conservation ◽  
Induced Mutations ◽  
Mutagenic Potential ◽  
E Coli ◽  
Antimutagenic Effect ◽  
Induced Mutagenesis

The aim of this work was to investigate the antimutagenic potential of monoterpenes from sage and basil in Escherichia coli. The mutagenic potential of monoterpenes was pre-screened with Salmonella/microsome reversion assay in strain TA100 and no mutagenic effect was detected. The antimutagenic potential against UV- 4NQO- and t-BOOH induced mutagenesis was evaluated in E. coli K12 and E. coli WP2 by reversion assays. The obtained results indicate that camphor and thujone reduce UV- and 4NQO-induced mutations; myrcene reduces t-BOOH-induced mutations, while eucalyptol and linalool reduce mutagenicity by all tested mutagens. Considering evolutionary conservation of DNA repair and antioxidative protection, the obtained results indicate that further antigenotoxicity studies should be undertaken in eukaryotes.

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