Mutagenesis in Escherichia coli : evidence for the mechanism of base change mutation by ultraviolet radiation in a strain deficient in excision-repair

1968 ◽  
Vol 171 (1023) ◽  
pp. 213-226 ◽  
Keyword(s):  
Escherichia Coli ◽  
Generation Time ◽  
Excision Repair ◽  
Indirect Evidence ◽  
Base Change ◽  
Pyrimidine Dimer ◽  
Mutagenic Action ◽  
Pyrimidine Dimers ◽  
Generation Times ◽  
Low Probability

The mutagenic action of u. v. radiation has been studied upon Escherichia coli WP2 try her growing exponentially at 37 °C. Although this strain is unable to excise pyrimidine dimers from its DNA it showed no detectable reduction in growth rate after exposure to a dose of u. v. (10 -6 J mm -2 ) calculated to produce several dozen pyrimidine dimers per chromosome. As judged by photoreversibility of mutations to prototrophy, dimers at mutable sites may persist for up to about 4 generation times after u. v. and may give rise to mutations with a low probability in each replication cycle during this period. The slow disappearance of dimers takes place whether or not DNA replication is inhibited and indirect evidence suggests that excision-repair may not be involved. Mutations are established (i. e. become non-photoreversible) only when DNA replication is taking place and are not expressible on unsupplemented medium until approximately one generation time after being established. It is suggested that the mutation is initially produced by the laying down of an incorrect base opposite a pyrimidine dimer at, or shortly after, replication; the mutation becomes transcribable only after a further replication gives rise to a duplex mutant in both strands. The observed segregation pattern strongly suggests that at this further replication the mutation is carried by both resulting daughter duplexes. This implies that the mutant strand initially produced opposite the dimer has a strong influence on the bases of both new strands laid down at the next replication.

scholarly journals Photolyases from Saccharomyces cerevisiae and Escherichia coli recognize common binding determinants in DNA containing pyrimidine dimers.

1989 ◽  
Vol 9 (11) ◽  
pp. 4777-4788 ◽  
Author(s):  
M Baer ◽  
G B Sancar
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Spectroscopic Studies ◽  
Pyrimidine Dimer ◽  
Nucleotide Sequence Analysis ◽  
Binding Motif ◽  
Pyrimidine Dimers ◽  
Nucleotide Excision

DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. The results of nucleotide sequence analysis and spectroscopic studies demonstrated that photolyases from Saccharomyces cerevisiae and Escherichia coli share 37% amino acid sequence homology and contain identical chromophores. Do the similarities between these two enzymes extend to their interactions with DNA containing pyrimidine dimers, or does the organization of DNA into nucleosomes in S. cerevisiae necessitate alternative or additional recognition determinants? To answer this question, we used chemical and enzymatic techniques to identify the contacts made on DNA by S. cerevisiae photolyase when it is bound to a pyrimidine dimer and compared these contacts with those made by E. coli photolyase and by a truncated derivative of the yeast enzyme when bound to the same substrate. We found evidence for a common set of interactions between the photolyases and specific phosphates in the backbones of both strands as well as for interactions with bases in both the major and minor grooves of dimer-containing DNA. Superimposed on this common pattern were significant differences in the contributions of specific contacts to the overall binding energy, in the interactions of the enzymes with groups on the complementary strand, and in the extent to which other DNA-binding proteins were excluded from the region around the dimer. These results provide strong evidence both for a conserved dimer-binding motif and for the evolution of new interactions that permit photolyases to also act as accessory proteins in nucleotide excision repair. The locations of the specific contacts made by the yeast enzyme indicate that the mechanism of nucleotide excision repair in this organism involves incision(s) at a distance from the pyrimidine dimer.

scholarly journals Photolyases from Saccharomyces cerevisiae and Escherichia coli recognize common binding determinants in DNA containing pyrimidine dimers

1989 ◽  
Vol 9 (11) ◽  
pp. 4777-4788
Author(s):  
M Baer ◽  
G B Sancar
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Spectroscopic Studies ◽  
Pyrimidine Dimer ◽  
Nucleotide Sequence Analysis ◽  
Binding Motif ◽  
Pyrimidine Dimers ◽  
Nucleotide Excision

DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. The results of nucleotide sequence analysis and spectroscopic studies demonstrated that photolyases from Saccharomyces cerevisiae and Escherichia coli share 37% amino acid sequence homology and contain identical chromophores. Do the similarities between these two enzymes extend to their interactions with DNA containing pyrimidine dimers, or does the organization of DNA into nucleosomes in S. cerevisiae necessitate alternative or additional recognition determinants? To answer this question, we used chemical and enzymatic techniques to identify the contacts made on DNA by S. cerevisiae photolyase when it is bound to a pyrimidine dimer and compared these contacts with those made by E. coli photolyase and by a truncated derivative of the yeast enzyme when bound to the same substrate. We found evidence for a common set of interactions between the photolyases and specific phosphates in the backbones of both strands as well as for interactions with bases in both the major and minor grooves of dimer-containing DNA. Superimposed on this common pattern were significant differences in the contributions of specific contacts to the overall binding energy, in the interactions of the enzymes with groups on the complementary strand, and in the extent to which other DNA-binding proteins were excluded from the region around the dimer. These results provide strong evidence both for a conserved dimer-binding motif and for the evolution of new interactions that permit photolyases to also act as accessory proteins in nucleotide excision repair. The locations of the specific contacts made by the yeast enzyme indicate that the mechanism of nucleotide excision repair in this organism involves incision(s) at a distance from the pyrimidine dimer.

scholarly journals The noncovalent complex between DNA and the bifunctional intercalator ditercalinium is a substrate for the UvrABC endonuclease of Escherichia coli

1989 ◽  
Vol 86 (17) ◽  
pp. 6557-6561 ◽  
Author(s):  
B Lambert ◽  
B K Jones ◽  
B P Roques ◽  
J B Le Pecq ◽  
A T Yeung
Keyword(s):  
Escherichia Coli ◽  
Half Life ◽  
Excision Repair ◽  
Noncovalent Complex ◽  
Dna Lesions ◽  
Pyrimidine Dimer ◽  
Stable Complex ◽  
Ligand Complex ◽  
Dna Complexes ◽  
Pyrimidine Dimers

We have demonstrated that the noncovalent complex formed between DNA and an antitumor bifunctional intercalator, ditercalinium, is recognized in vitro as bulky covalent DNA lesions by the purified Escherichia coli UvrABC endonuclease. It was established that no covalent drug-DNA adduct was formed during the incubation of the drug with DNA or during subsequent incubation with the UvrAB proteins. The nucleoprotein-ditercalinium complexes appear different from those generated by repair of pyrimidine dimers. The UvrA protein is able to form a stable complex with ditercalinium-intercalated DNA in the presence of ATP, whereas both UvrA and UvrB proteins are required to form a stable complex with pyrimidine dimer-containing DNA. The apparent half-life of the UvrA- and UvrAB-ditercalinium-DNA complexes following removal of free ditercalinium is 5 min. However, if the free ditercalinium concentration is maintained to allow the intercalation of one molecule of ditercalinium per 3000 base pairs, the half-life of the UvrA- or UvrAB-ditercalinium-DNA complex is 50 min, comparable to that of the complex of UvrAB proteins formed with pyrimidine dimer-containing DNA. UvrABC endonuclease incises ditercalinium-intercalated DNA as efficiently as pyrimidine dimer-containing DNA. However, unlike repair of pyrimidine dimers, the incision reaction is strongly favored by the supercoiling of the DNA substrate. Because UvrA- or UvrAB-ditercalinium-DNA complexes can be formed with relaxed DNA without leading to a subsequent incision reaction, these apparently dead-end nucleoprotein complexes may become lesions in themselves resulting in the cytotoxicity of ditercalinium. Our results show that binding of excision repair proteins to a noncovalent DNA-ligand complex may lead to cell toxicity.

scholarly journals Repair of abasic sites by mammalian cell extracts

1994 ◽  
Vol 304 (3) ◽  
pp. 699-705 ◽  
Author(s):  
G Frosina ◽  
P Fortini ◽  
O Rossi ◽  
F Carrozzino ◽  
A Abbondandolo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Excision Repair ◽  
Chinese Hamster ◽  
Pyrimidine Dimer ◽  
Repair Synthesis ◽  
Repair Patch ◽  
Cell Extracts ◽  
Pyrimidine Dimers ◽  
Ap Sites ◽  
Ap Site

Hamster cell extracts that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers, were used to study the repair of apurinic/apyrimidinic (AP) sites and methoxyamine (MX)-modified AP sites. Plasmid molecules were heat-treated at pH 5 and incubated with MX when required. The amount of damage introduced ranged from 0.2 to 0.9 AP sites/kb. Extracts were prepared from the Chinese hamster ovary CHO-9 cell line and from its derivative, 43-3B clone which is mutated in the nucleotide excision repair (NER) ERCC1 gene. AP and MX-AP sites stimulated repair synthesis by CHO-9 cell extracts. The level of synthesis correlated with the number of lesions and was of similar magnitude to the repair stimulated by 4.3 u.v. photoproducts/kb. Repair of AP and MX-AP sites was faster than the repair of u.v. damage and was independent of ERCC1 gene product. The high level of repair replication was due to a very efficient and rapid incision of plasmids carrying AP or MX-AP sites, performed by abundant AP endonucleases present in the extract. The calculated average repair patch sizes were: 7 nucleotides per AP site; 10 nucleotides per MX-AP site; 28 nucleotides per (6-4) u.v. photoproduct or cyclobutane pyrimidine dimer. The data indicate that AP and MX-AP sites are very efficiently repaired by base-excision repair in mammalian cells and suggest that MX-AP sites may also be processed via alternative repair mechanisms.

The RAD7 and RAD16 genes, which are essential for pyrimidine dimer removal from the silent mating type loci, are also required for repair of the nontranscribed strand of an active gene in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (9) ◽  
pp. 6135-6142
Author(s):  
R Verhage ◽  
A M Zeeman ◽  
N de Groot ◽  
F Gleig ◽  
D D Bang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Excision Repair ◽  
Complementation Group ◽  
Pyrimidine Dimer ◽  
Gene Products ◽  
Pyrimidine Dimers ◽  
Nucleotide Excision ◽  
Active Genes ◽  
Silent Mating Type Loci

The rad16 mutant of Saccharomyces cerevisiae was previously shown to be impaired in removal of UV-induced pyrimidine dimers from the silent mating-type loci (D. D. Bang, R. A. Verhage, N. Goosen, J. Brouwer, and P. van de Putte, Nucleic Acids Res. 20:3925-3931, 1992). Here we show that rad7 as well as rad7 rad16 double mutants have the same repair phenotype, indicating that the RAD7 and RAD16 gene products might operate in the same nucleotide excision repair subpathway. Dimer removal from the genome overall is essentially incomplete in these mutants, leaving about 20 to 30% of the DNA unrepaired. Repair analysis of the transcribed RPB2 gene shows that the nontranscribed strand is not repaired at all in rad7 and rad16 mutants, whereas the transcribed strand is repaired in these mutants at a fast rate similar to that in RAD+ cells. When the results obtained with the RPB2 gene can be generalized, the RAD7 and RAD16 proteins not only are essential for repair of silenced regions but also function in repair of nontranscribed strands of active genes in S. cerevisiae. The phenotype of rad7 and rad16 mutants closely resembles that of human xeroderma pigmentosum complementation group C (XP-C) cells, suggesting that RAD7 and RAD16 in S. cerevisiae function in the same pathway as the XPC gene in human cells. RAD4, which on the basis of sequence homology has been proposed to be the yeast XPC counterpart, seems to be involved in repair of both inactive and active yeast DNA, challenging the hypothesis that RAD4 and XPC are functional homologs.

scholarly journals The Roles of Several Residues ofEscherichia coliDNA Photolyase in the Highly Efficient Photo-Repair of Cyclobutane Pyrimidine Dimers

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Lei Xu ◽  
Guoping Zhu
Keyword(s):  
Escherichia Coli ◽  
Energy Source ◽  
Pyrimidine Dimer ◽  
Cyclobutane Pyrimidine Dimer ◽  
Model Compounds ◽  
Cyclobutane Pyrimidine Dimers ◽  
Dna Photolyase ◽  
Pyrimidine Dimers ◽  
Repair Efficiency ◽  
Very High

Escherichia coliDNA photolyase is an enzyme that repairs the major kind of UV-induced lesions, cyclobutane pyrimidine dimer (CPD) in DNA utilizing 350–450 nm light as energy source. The enzyme has very high photo-repair efficiency (the quantum yield of the reaction is ~0.85), which is significantly greater than many model compounds that mimic photolyase. This suggests that some residues of the protein play important roles in the photo-repair of CPD. In this paper, we have focused on several residues discussed their roles in catalysis by reviewing the existing literature and some hypotheses.

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 Mutagenic Action of Phaseolinone, a Mycotoxin Isolated from Macrophomina phaseolina

1987 ◽  
Vol 40 (4) ◽  
pp. 349 ◽  
Author(s):  
Gautam Bhattacharya ◽  
Tarun K Dhar ◽  
F Kathleen Bhattacharya ◽  
Kazi A I Siddiqui
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Excision Repair ◽  
Mutagenic Activity ◽  
Covalent Binding ◽  
Macrophomina Phaseolina ◽  
Side Chain ◽  
Mutagenic Action ◽  
Hydroxy Groups

Phaseolinone was mutagenic to excision-repair-deficient strains of Escherichia coli WP2 and also to Salmonella typhimurium TA100. The repair test was indicative of covalent binding of the toxin to DNA. The side-chain epoxide and the hydroxy groups of the molecule were found to be essential for mutagenic activity.

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