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1.
Mutat Res ; 759: 1-8, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24361397

ABSTRACT

The spontaneous incidence of chloramphenicol (Cam) resistant mutant bacteria is at least ten-fold higher in cultures of enterohemorrhagic Escherichia coli O157:H7 strain EDL933 than in E. coli K-12. It is at least 100-fold higher in the dam (DNA adenine methyltransferase) derivative of EDL933, compared to the dam strain of E. coli K-12, thereby preventing the use of Cam resistance as a marker in gene replacement technology. Genome sequencing of Cam-resistant isolates of EDL933 and its dam derivatives showed that the marR (multiple antibiotic resistance) gene was mutated in every case but not in the Cam-sensitive parental strains. As expected from mutation in the marR gene, the Cam-resistant bacteria were also found to be resistant to tetracycline and nalidixic acid. The marR gene in strain EDL933 is annotated as a shorter open reading frame than that in E. coli K-12 but the longer marR(+) open reading frame was more efficient at complementing the marR antibiotic-resistance phenotype of strain EDL933. Beta-lactamase-tolerant derivatives were present at frequencies 10-100 times greater in cultures of marR derivatives of strain EDL933 than the parent strain. Spontaneous mutation frequency to rifampicin, spectinomycin and streptomycin resistance was the same in E. coli O157:H7 and E. coli K-12 strains.


Subject(s)
Drug Resistance, Multiple, Bacterial , Escherichia coli O157/drug effects , Carbenicillin/pharmacology , Chloramphenicol/pharmacology , Escherichia coli O157/genetics , Escherichia coli Proteins/genetics , Genetic Complementation Test , Microbial Sensitivity Tests , Mutation , Repressor Proteins/genetics
2.
F1000Res ; 2: 7, 2013.
Article in English | MEDLINE | ID: mdl-24358856

ABSTRACT

Genetic manipulation in enterohemorrhagic E. coli O157:H7 is currently restricted to recombineering, a method that utilizes the recombination system of bacteriophage lambda, to introduce gene replacements and base changes inter alia into the genome. Bacteriophage 933W is a prophage in E. coli O157:H7 strain EDL933, which encodes the genes ( stx2AB) for the production of Shiga toxin which is the basis for the potentially fatal Hemolytic Uremic Syndrome in infected humans. We replaced the stx2AB genes with a kanamycin cassette using recombineering. After induction of the prophage by ultra-violet light, we found that bacteriophage lysates were capable of transducing to wildtype, point mutations in the lactose, arabinose and maltose genes. The lysates could also transduce tetracycline resistant cassettes. Bacteriophage 933W is also efficient at transducing markers in E. coli K-12. Co-transduction experiments indicated that the maximal amount of transferred DNA was likely the size of the bacteriophage genome, 61 kB. All tested transductants, in both E. coli K-12 and O157:H7, were kanamycin-sensitive indicating that the transducing particles contained host DNA.

3.
Nucleic Acids Res ; 41(17): 8166-81, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23821665

ABSTRACT

The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair.


Subject(s)
Base Pair Mismatch , DNA/chemistry , Escherichia coli Proteins/chemistry , MutS DNA Mismatch-Binding Protein/chemistry , Adenosine Triphosphate/metabolism , DNA/metabolism , Escherichia coli Proteins/metabolism , Models, Molecular , MutS DNA Mismatch-Binding Protein/metabolism , Protein Binding , Protein Multimerization , Protein Structure, Tertiary
4.
PLoS One ; 8(3): e60519, 2013.
Article in English | MEDLINE | ID: mdl-23544153

ABSTRACT

The nematode Caenorhabditis elegans has in recent years been proven to be a powerful in vivo model for testing antimicrobial compounds. We report here that the alkaloid compound Harmane (2-methyl-ß-carboline) increases the lifespan of nematodes infected with a human pathogen, the Shiga toxin-producing Escherichia coli O157:H7 strain EDL933 and several other bacterial pathogens. This was shown to be unrelated to the weak antibiotic effect of Harmane. Using GFP-expressing E. coli EDL933, we showed that Harmane does not lower the colonization burden in the nematodes. We also found that the expression of the putative immune effector gene F35E12.5 was up-regulated in response to Harmane treatment. This indicates that Harmane stimulates the innate immune response of the nematode; thereby increasing its lifespan during bacterial infection. Expression of F35E12.5 is predominantly regulated through the p38 MAPK pathway; however, intriguingly the lifespan extension resulting from Harmane was higher in p38 MAPK-deficient nematodes. This indicates that Harmane has a complex effect on the innate immune system of C. elegans. Harmane could therefore be a useful tool in the further research into C. elegans immunity. Since the innate immunity of C. elegans has a high degree of evolutionary conservation, drugs such as Harmane could also be possible alternatives to classic antibiotics. The C. elegans model could prove to be useful for selection and development of such drugs.


Subject(s)
Bacterial Infections/immunology , Caenorhabditis elegans/immunology , Caenorhabditis elegans/microbiology , Harmine/analogs & derivatives , Immunity, Innate/drug effects , Longevity/drug effects , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Infections/pathology , Caenorhabditis elegans/drug effects , Caenorhabditis elegans/physiology , Caenorhabditis elegans Proteins/metabolism , Colony Count, Microbial , Escherichia coli/drug effects , Escherichia coli/growth & development , Harmine/pharmacology , Hot Temperature , Humans , Immunity, Innate/genetics , Intestines/drug effects , Intestines/microbiology , Intestines/pathology , Longevity/immunology , MAP Kinase Kinase 4/metabolism , Receptor, Insulin/metabolism , Signal Transduction/drug effects
5.
PLoS One ; 7(3): e33256, 2012.
Article in English | MEDLINE | ID: mdl-22438905

ABSTRACT

Bleomycin (BLM) is a glycopeptide antibiotic and anti-tumor agent that targets primarily the furanose rings of DNA and in the presence of ferrous ions produces oxidative damage and DNA strand breaks. Escherichia coli cells growing in broth medium and exposed to low concentrations of BLM contain double-strand breaks and require homologous recombination to survive. To a lesser extent, the cells also require the abasic (AP) endonucleases associated with base excision repair, presumably to repair oxidative damage. As expected, there is strong induction of the SOS system in treated cells. In contrast, E. coli cells growing in glucose or glycerol minimal medium are resistant to the lethal action of BLM and do not require either homologous recombination functions or AP-endonucleases for survival. DNA ligase activity, however, is needed for cells growing in minimal medium to resist the lethal effects of BLM. There is weak SOS induction in such treated cells.


Subject(s)
Bleomycin/pharmacology , Escherichia coli K12/drug effects , Antibiotics, Antineoplastic/pharmacology , Bacterial Proteins/genetics , Culture Media , DNA Breaks, Double-Stranded , DNA Ligases/metabolism , DNA Restriction Enzymes/genetics , DNA, Bacterial/drug effects , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Drug Resistance, Bacterial , Escherichia coli K12/genetics , Escherichia coli K12/metabolism , Escherichia coli Proteins/genetics , Genes, Bacterial , Mutation , Oligonucleotide Array Sequence Analysis , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , Recombinational DNA Repair/genetics , SOS Response, Genetics/drug effects
6.
F1000 Biol Rep ; 2: 56, 2010 Jul 22.
Article in English | MEDLINE | ID: mdl-20711416

ABSTRACT

The expression of Beta, the single-stranded annealing protein (SSAP) of bacteriophage lambda in Escherichia coli promotes high levels of oligonucleotide (oligo)-mediated mutagenesis and offers a quick way to create single or multiple base pair insertions, deletions, or substitutions in the bacterial chromosome. High rates of mutagenesis can be obtained by the use of mismatch repair (MMR)-resistant mismatches or MMR-deficient hosts, which allow for the isolation of unselected mutations. It has recently become clear that many bacteria can be mutagenized with oligos in the absence of any SSAP expression, albeit at a much lower frequency. Studies have shown that inactivation or inhibition of single-stranded DNA (ssDNA) exonucleases in vivo increases the rate of SSAP-independent oligo-mediated mutagenesis. These results suggest that lambda Beta, in addition to its role in annealing the oligo to ssDNA regions of the replication fork, promotes high rates of oligo-mediated mutagenesis by protecting the oligo from destruction by host ssDNA exonucleases.

7.
Mutat Res ; 705(2): 71-76, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20471491

ABSTRACT

Mutator strains of Escherichia coli have been used to define mechanisms that account for the high fidelity of chromosome duplication and chromosome stability. Mutant strains defective in post-replicative mismatch repair display a strong mutator phenotype consistent with a role for correction of mismatches arising from replication errors. Inactivation of the gene (dam) encoding DNA adenine methyltransferase results in a mutator phenotype consistent with a role for DNA methylation in strand discrimination during mismatch repair. This review gives a personal perspective on the discovery of dam mutants in E. coli and their relationship to mismatch repair and mutator phenotypes.


Subject(s)
DNA Methylation , DNA, Bacterial/metabolism , Escherichia coli/genetics , Genes, Bacterial , Methylnitronitrosoguanidine/pharmacology , Mutation , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics , DNA Mismatch Repair , Recombination, Genetic
8.
FEMS Microbiol Rev ; 33(3): 488-503, 2009 May.
Article in English | MEDLINE | ID: mdl-19175412

ABSTRACT

The DNA adenine methyltransferase (Dam methylase) of Gammaproteobacteria and the cell cycle-regulated methyltransferase (CcrM) methylase of Alphaproteobacteria catalyze an identical reaction (methylation of adenosine moieties using S-adenosyl-methionine as a methyl donor) at similar DNA targets (GATC and GANTC, respectively). Dam and CcrM are of independent evolutionary origin. Each may have evolved from an ancestral restriction-modification system that lost its restriction component, leaving an 'orphan' methylase devoted solely to epigenetic genome modification. The formation of 6-methyladenine reduces the thermodynamic stability of DNA and changes DNA curvature. As a consequence, the methylation state of specific adenosine moieties can affect DNA-protein interactions. Well-known examples include binding of the replication initiation complex to the methylated oriC, recognition of hemimethylated GATCs in newly replicated DNA by the MutHLS mismatch repair complex, and discrimination of methylation states in promoters and regulatory DNA motifs by RNA polymerase and transcription factors. In recent years, Dam and CcrM have been shown to play roles in host-pathogen interactions. These roles are diverse and have only partially been understood. Especially intriguing is the evidence that Dam methylation regulates virulence genes in Escherichia coli, Salmonella, and Yersinia at the posttranscriptional level.


Subject(s)
Adenine/metabolism , Bacterial Physiological Phenomena , DNA Methylation , DNA Mismatch Repair , DNA, Bacterial/metabolism , Gene Expression Regulation, Bacterial , Host-Pathogen Interactions , DNA Modification Methylases/metabolism
9.
PLoS One ; 3(8): e2984, 2008 Aug 20.
Article in English | MEDLINE | ID: mdl-18714349

ABSTRACT

BACKGROUND: Initiation of chromosome replication in E. coli requires the DnaA and DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to synchronize cell populations. METHODOLOGY/PRINCIPAL FINDINGS: DNA microarrays were used to measure mRNA steady-state levels in initiation-deficient dnaA46 and dnaC2 bacteria at permissive and non-permissive temperatures and their expression profiles were compared to MG1655 wildtype cells. For both mutants there was altered expression of genes involved in nucleotide biosynthesis at the non-permissive temperature. Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes. Induction of the SOS regulon was observed in dnaC2 cells at 38 degrees C and 42 degrees C. Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation. CONCLUSION/SIGNIFICANCE: We suggest that in dnaC2 cells the SOS response is triggered by persistent open-complex formation at oriC and/or by arrested forks that require DnaC for replication restart.


Subject(s)
Escherichia coli K12/genetics , Escherichia coli Proteins/antagonists & inhibitors , Nucleotides/genetics , SOS Response, Genetics , Bacterial Proteins/genetics , Chromosomes, Bacterial/genetics , DNA Replication/genetics , DNA-Binding Proteins/genetics , Escherichia coli K12/metabolism , Escherichia coli Proteins/genetics , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Genes, Bacterial , Genes, Lethal , Heat-Shock Proteins/genetics , Nucleotides/biosynthesis , Oligonucleotide Array Sequence Analysis , RNA, Bacterial/genetics , RNA, Bacterial/isolation & purification , RNA, Messenger/genetics , Thermodynamics , Transcription, Genetic
10.
J Bacteriol ; 189(11): 4325-7, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17400740

ABSTRACT

The Caulobacter crescentus DNA adenine methyltransferase CcrM and its homologs in the alpha-Proteobacteria are essential for viability. CcrM is 34% identical to the yhdJ gene products of Escherichia coli and Salmonella enterica. This study provides evidence that the E. coli yhdJ gene encodes a DNA adenine methyltransferase. In contrast to an earlier report, however, we show that yhdJ is not an essential gene in either E. coli or S. enterica.


Subject(s)
DNA Modification Methylases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Salmonella enterica/enzymology , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Binding Sites/genetics , Catalytic Domain/genetics , DNA Methylation , DNA Modification Methylases/genetics , Deoxyribonucleases, Type II Site-Specific/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Plasmids/genetics , Plasmids/metabolism , Salmonella enterica/genetics , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics
11.
Genes Dev ; 20(15): 2121-34, 2006 Aug 01.
Article in English | MEDLINE | ID: mdl-16882985

ABSTRACT

Initiation of DNA replication in Eschericia coli requires the ATP-bound form of the DnaA protein. The conversion of DnaA-ATP to DnaA-ADP is facilitated by a complex of DnaA, Hda (homologous to DnaA), and DNA-loaded beta-clamp proteins in a process termed RIDA (regulatory inactivation of DnaA). Hda-deficient cells initiate replication at each origin mainly once per cell cycle, and the rare reinitiation events never coincide with the end of the origin sequestration period. Therefore, RIDA is not the predominant mechanism to prevent immediate reinitiation from oriC. The cellular level of Hda correlated directly with dnaA gene expression such that Hda deficiency led to reduced dnaA gene expression, and overproduction of Hda led to DnaA overproduction. Hda-deficient cells were very sensitive to variations in the cellular level of DnaA, and DnaA overproduction led to uncontrolled initiation of replication from oriC, causing severe growth retardation or cell death. Based on these observations, we propose that both RIDA and dnaA gene autoregulation are required as homeostatic mechanisms to ensure that initiation of replication occurs at the same time relative to cell mass in each cell cycle.


Subject(s)
Adenosine Triphosphatases/metabolism , Bacterial Proteins/metabolism , Chromosomes, Bacterial , DNA-Binding Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli , Gene Expression Regulation, Bacterial , Homeostasis , Adenosine Triphosphate/metabolism , Bacterial Proteins/genetics , Cell Death , DNA Replication , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Replication Origin , Trans-Activators
12.
J Biol Chem ; 281(20): 14340-9, 2006 May 19.
Article in English | MEDLINE | ID: mdl-16546997

ABSTRACT

The MutL and MutS proteins are the central components of the DNA repair machinery that corrects mismatches generated by DNA polymerases during synthesis. We find that MutL interacts directly with the beta sliding clamp, a ring-shaped dimeric protein that confers processivity to DNA polymerases by tethering them to their substrates. Interestingly, the interaction of MutL with beta only occurs in the presence of single-stranded DNA. We find that the interaction occurs via a loop in MutL near the ATP-binding site. The binding site of MutL on beta locates to the hydrophobic pocket between domains two and three of the clamp. Site-specific replacement of two residues in MutL diminished interaction with beta without disrupting MutL function with helicase II. In vivo studies reveal that this mutant MutL is no longer functional in mismatch repair. In addition, the human MLH1 has a close match to the proliferating cell nuclear antigen clamp binding motif in the region that corresponds to the beta interaction site in Escherichia coli MutL, and a peptide corresponding to this site binds proliferating cell nuclear antigen. The current report also examines in detail the interaction of beta with MutS. We find that two distinct regions of MutS interact with beta. One is located near the C terminus and the other is close to the N terminus, within the mismatch binding domain. Complementation studies using genes encoding different MutS mutants reveal that the N-terminal beta interaction motif on MutS is essential for activity in vivo, but the C-terminal interaction site for beta is not. In light of these results, we propose roles for the beta clamp in orchestrating the sequence of events that lead to mismatch repair in the cell.


Subject(s)
Adenosine Triphosphatases/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/metabolism , MutS DNA Mismatch-Binding Protein/chemistry , Adaptor Proteins, Signal Transducing , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Carrier Proteins/chemistry , DNA Repair , DNA, Single-Stranded/chemistry , Dose-Response Relationship, Drug , Escherichia coli Proteins/metabolism , Genetic Complementation Test , Humans , Models, Molecular , Molecular Sequence Data , MutL Protein Homolog 1 , MutL Proteins , MutS DNA Mismatch-Binding Protein/metabolism , Nuclear Proteins/chemistry , Protein Binding , Sequence Homology, Amino Acid
13.
Curr Opin Microbiol ; 8(2): 154-60, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15802246

ABSTRACT

GATC sequences in Escherichia coli DNA are methylated at the adenine residue by DNA adenine methyltransferase (DamMT). These methylated residues and/or the level of DamMT can influence cellular functions such as gene transcription, DNA mismatch repair, initiation of chromosome replication and nucleoid structure. In certain bacteria, unlike E. coli, DamMT is essential for viability perhaps owing to its role in chromosome replication. DamMT has also been implicated as a virulence factor in bacterial pathogenesis. The origin and phylogeny of DamMT, based on sequenced genomes, has been deduced.


Subject(s)
Bacterial Physiological Phenomena , DNA Methylation , DNA, Bacterial/metabolism , Gene Expression Regulation, Bacterial , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics , Site-Specific DNA-Methyltransferase (Adenine-Specific)/physiology , Escherichia coli Proteins
14.
Mutat Res ; 554(1-2): 149-57, 2004 Oct 04.
Article in English | MEDLINE | ID: mdl-15450413

ABSTRACT

The sensitivity of a panel of DNA repair-defective bacterial strains to BLM was investigated. Escherichia coli recA cells were far more sensitive than were uvrA, dam-3, and mutM mutY strains, underscoring the importance of RecA to survival. Strains recBCD and recN, which lack proteins required for double strand break (DSB) repair, were highly sensitive to BLM, while recF cells were not. The requirement for DSB-specific enzymes supports the hypothesis that DSBs are the primary cause of bleomycin cytotoxicity. The acute sensitivity of recN cells was comparable to that of recA, implying a central role for the RecN protein in BLM lesion repair. The Holliday junction processing enzymes RecG and RuvC were both required for BLM survival. The recG ruvC double mutant was no more sensitive than either mutation alone, suggesting that both enzymes participate in the same pathway. Surprisingly, ruvAB cells were no more sensitive than wildtype, implying that RuvC is able to perform its role without RuvAB. This observation contrasts with current models of recombination in which RuvA, B, and C function as a single complex. The most straightforward explanation of these results is that DSB repair involves a structure that serves as a good substrate for RecG, and not RuvAB.


Subject(s)
Bacterial Proteins/physiology , Bleomycin/toxicity , DNA Restriction Enzymes/physiology , Escherichia coli Proteins/physiology , Escherichia coli/drug effects , Mutagens/toxicity , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/growth & development , Recombination, Genetic
15.
Proc Natl Acad Sci U S A ; 100(8): 4672-7, 2003 Apr 15.
Article in English | MEDLINE | ID: mdl-12682301

ABSTRACT

High-density oligonucleotide arrays were used to monitor global transcription patterns in Escherichia coli with various levels of Dam and SeqA proteins. Cells lacking Dam methyltransferase showed a modest increase in transcription of the genes belonging to the SOS regulon. Bacteria devoid of the SeqA protein, which preferentially binds hemimethylated DNA, were found to have a transcriptional profile almost identical to WT bacteria overexpressing Dam methyltransferase. The latter two strains differed from WT in two ways. First, the origin proximal genes were transcribed with increased frequency due to increased gene dosage. Second, chromosomal domains of high transcriptional activity alternate with regions of low activity, and our results indicate that the activity in each domain is modulated in the same way by SeqA deficiency or Dam overproduction. We suggest that the methylation status of the cell is an important factor in forming and/or maintaining chromosome structure.


Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli/genetics , Genes, Bacterial , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics , Transcription Factors/genetics , Bacterial Outer Membrane Proteins , Chromosomes, Bacterial/genetics , DNA Methylation , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA-Binding Proteins , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Oligonucleotide Array Sequence Analysis , SOS Response, Genetics
16.
Nucleic Acids Res ; 31(7): 1805-12, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12654995

ABSTRACT

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.


Subject(s)
DNA Restriction Enzymes/classification , Methyltransferases/classification , Terminology as Topic , Base Sequence , Binding Sites , DNA/genetics , DNA/metabolism , DNA Restriction Enzymes/genetics , DNA Restriction Enzymes/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism
17.
J Bacteriol ; 184(13): 3501-7, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12057944

ABSTRACT

Nitric oxide (NO*) is involved in neurotransmission, inflammation, and many other biological processes. Exposure of cells to NO* leads to DNA damage, including formation of deaminated and oxidized bases. Apurinic/apyrimidinic (AP) endonuclease-deficient cells are sensitive to NO* toxicity, which indicates that base excision repair (BER) intermediates are being generated. Here, we show that AP endonuclease-deficient cells can be protected from NO* toxicity by inactivation of the uracil (Ung) or formamidopyrimidine (Fpg) DNA glycosylases but not by inactivation of a 3-methyladenine (AlkA) DNA glycosylase. These results suggest that Ung and Fpg remove nontoxic NO*-induced base damage to create BER intermediates that are toxic if they are not processed by AP endonucleases. Our next goal was to learn how Ung and Fpg affect susceptibility to homologous recombination. The RecBCD complex is critical for repair of double-strand breaks via homologous recombination. When both Ung and Fpg were inactivated in recBCD cells, survival was significantly enhanced. We infer that both Ung and Fpg create substrates for recombinational repair, which is consistent with the observation that disrupting ung and fpg suppressed NO*-induced recombination. Taken together, a picture emerges in which the action of DNA glycosylases on NO*-induced base damage results in the accumulation of BER intermediates, which in turn can induce homologous recombination. These studies shed light on the underlying mechanism of NO*-induced homologous recombination.


Subject(s)
Escherichia coli/genetics , N-Glycosyl Hydrolases/metabolism , Nitric Oxide/metabolism , Recombination, Genetic , Carbon-Oxygen Lyases/genetics , Carbon-Oxygen Lyases/metabolism , DNA Damage/drug effects , DNA Glycosylases , DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase , DNA-Directed DNA Polymerase , DNA-Formamidopyrimidine Glycosylase , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Exodeoxyribonuclease V , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Mutation , N-Glycosyl Hydrolases/genetics , Nitric Oxide/pharmacology , Uracil-DNA Glycosidase
18.
J Biol Chem ; 277(2): 1255-60, 2002 Jan 11.
Article in English | MEDLINE | ID: mdl-11705991

ABSTRACT

Loss of mismatch repair leads to tumor resistance by desensitizing cells to specific DNA-damaging agents, including the anticancer drug cisplatin. Cisplatin analogs with a diamminocyclohexane (DACH) carrier ligand, such as oxaliplatin and Pt(DACH)Cl(2), do not elicit resistance in mismatch repair-deficient cells and therefore present promising therapeutic agents. This study compared the interactions of the purified Escherichia coli mismatch repair protein MutS with DNA modified to contain cisplatin and DACH adducts. MutS recognized the cisplatin-modified DNA with 2-fold higher affinity in comparison to the DACH-modified DNA. ADP stimulated the binding of MutS to cisplatin-modified DNA, whereas it had no effect on the MutS interaction with DNA modified by DACH or EN adducts. In parallel cytotoxicity experiments, methylation-deficient E. coli dam mutants were 2-fold more sensitive to cisplatin than DACH compounds. A panel of recombination-deficient mutants showed striking sensitivity to both compounds, indicating that both types of adducts are strong replication blocks. The differential affinity of MutS for DNA modified with the different platinum analogs could provide the molecular basis for the distinctive cellular responses to cisplatin and oxaliplatin.


Subject(s)
Adenosine Triphosphatases , Bacterial Proteins/metabolism , Cisplatin/metabolism , DNA Adducts/metabolism , DNA-Binding Proteins , Escherichia coli Proteins , Organoplatinum Compounds/metabolism , Antineoplastic Agents/metabolism , Base Pair Mismatch , Cell Survival , DNA Repair , Escherichia coli/genetics , Escherichia coli/physiology , Molecular Structure , MutS DNA Mismatch-Binding Protein , Organoplatinum Compounds/chemistry , Oxaliplatin
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