A region of the Ada DNA-repair protein required for the activation of ada transcription is not necessary for activation of alkA (original) (raw)
Related papers
Journal of Molecular Biology, 1988
Ada protein plays a central role in the regulatory synthesis of DNA repair enzymes, following exposure of Escherichia coli to alkylating agents. Methyl groups of alkylated DNA are transferred to Ada protein by its own methyltransferase activity and the methylated Ada protein then acts as a positive regulator to overproduce the ada and related gene products. To elucidate regulatory mechanisms for the expression of the ada gene by its own product, we analyzed the ada promoter region by random and site-directed mutagenesis. A series of deletion analyses revealed that a sequence up to 53 nucleotides upstream from the transcription initiation site is required for the controlled expression of the ada gene. Libraries of base substitution mutants were constructed by synthesizing oligonucleotides corresponding to the ada promoter region in the presence of a small amount of all possible sets of nucleotides. Internal deletion and insertion mutants were also constructed with the use of synthetic oligonucleotides. Using these mutants, the-10 and the-35 boxes of the promoter as well as the ada regulatory sequence were identified, the latter being an eightnucleotide sequence, AAAGCGCA. A six-nucleotide stretch between the regulatory sequence and the-35 box, also affected levels of expression of the gene. When the promoter DNAs derived from wild type or base substitution mutants that showed normal expression in vivo were used as templates for transcription in vitro, the adaspecific RNA was formed in the presence of a methylated form of Ada protein. With the DNAs derived from mutants of defective type as templates, no or relatively small amounts of the RNA were synthesized. Some base substitution mutants showed a constitutive expression of the gene in vivo, but this observation did not reconcile with findings in experiments in vitro.
J Mol Biol, 1988
Ada protein plays a central role in the regulatory synthesis of DNA repair enzymes, following exposure of Escherichia coli to alkylating agents. Methyl groups of alkylated DNA are transferred to Ada protein by its own methyltransferase activity and the methylated Ada protein then acts as a positive regulator to overproduce the ada and related gene products. To elucidate regulatory mechanisms for the expression of the ada gene by its own product, we analyzed the ada promoter region by random and site-directed mutagenesis. A series of deletion analyses revealed that a sequence up to 53 nucleotides upstream from the transcription initiation site is required for the controlled expression of the ada gene. Libraries of base substitution mutants were constructed by synthesizing oligonucleotides corresponding to the ada promoter region in the presence of a small amount of all possible sets of nucleotides. Internal deletion and insertion mutants were also constructed with the use of synthetic oligonucleotides. Using these mutants, the-10 and the-35 boxes of the promoter as well as the ada regulatory sequence were identified, the latter being an eightnucleotide sequence, AAAGCGCA. A six-nucleotide stretch between the regulatory sequence and the-35 box, also affected levels of expression of the gene. When the promoter DNAs derived from wild type or base substitution mutants that showed normal expression in vivo were used as templates for transcription in vitro, the adaspecific RNA was formed in the presence of a methylated form of Ada protein. With the DNAs derived from mutants of defective type as templates, no or relatively small amounts of the RNA were synthesized. Some base substitution mutants showed a constitutive expression of the gene in vivo, but this observation did not reconcile with findings in experiments in vitro.
Expression of the ada gene of Escherichia coli in response to alkylating agents
Journal of Molecular Biology, 1988
Ada protein plays a central role in the regulatory synthesis of DNA repair enzymes, following exposure of Escherichia coli to alkylating agents. Methyl groups of alkylated DNA are transferred to Ada protein by its own methyltransferase activity and the methylated Ada protein then acts as a positive regulator to overproduce the ada and related gene products. To elucidate regulatory mechanisms for the expression of the ada gene by its own product, we analyzed the ada promoter region by random and site-directed mutagenesis. A series of deletion analyses revealed that a sequence up to 53 nucleotides upstream from the transcription initiation site is required for the controlled expression of the ada gene. Libraries of base substitution mutants were constructed by synthesizing oligonucleotides corresponding to the ada promoter region in the presence of a small amount of all possible sets of nucleotides. Internal deletion and insertion mutants were also constructed with the use of synthetic oligonucleotides. Using these mutants, the-10 and the-35 boxes of the promoter as well as the ada regulatory sequence were identified, the latter being an eightnucleotide sequence, AAAGCGCA. A six-nucleotide stretch between the regulatory sequence and the-35 box, also affected levels of expression of the gene. When the promoter DNAs derived from wild type or base substitution mutants that showed normal expression in vivo were used as templates for transcription in vitro, the adaspecific RNA was formed in the presence of a methylated form of Ada protein. With the DNAs derived from mutants of defective type as templates, no or relatively small amounts of the RNA were synthesized. Some base substitution mutants showed a constitutive expression of the gene in vivo, but this observation did not reconcile with findings in experiments in vitro.
Ada response - a strategy for repair of alkylated DNA in bacteria
FEMS Microbiology Letters, 2014
Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N 3 -methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N 1 -methyladenine (1meA) and N 3 -methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O 6 -methylguanine (O 6 meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA.
Regulatory elements for expression of the alkA gene in response to alkylating agents
Molecular & general genetics : MGG, 1992
Expression of the alkA gene in Escherichia coli is controlled by Ada protein, which binds to a specific region of the alkA promoter and enhances further binding of RNA polymerase holoenzyme to the complex. To determine the sequence recognized by the Ada protein, we introduced various base substitutions into the promoter region of alkA and examined their effects on expression of the gene, both in vivo and in vitro. Base changes within the sequence AAAGCAAA, located between positions -41 and -34 from the transcription initiation site, greatly decreased the frequencies of initiation of transcription. In footprinting experiments, the region containing this sequence was protected by the Ada protein and base changes within this sequence led to failure of binding of Ada protein to the promoter. It is likely that the Ada protein recognizes the AAAGCAAA sequence in the alkA promoter and binds to the region containing the sequence, thereby allowing ready access of RNA polymerase to the promot...
Nucleic Acids Research, 1986
The activated Ada protein triggers expression of DNA repair genes in Escherichia coli in response to alkylation damage. Ada also osesses two distinct suicide alkyltransferase activities, for 0-alkylguanines and for alkyl phosphotriesters in DNA. The mutant Ada3 and Ada5 transferases repair 06-methylguanine in DNA 20 and 3000 times more slowly, respectively, than the wild-type Ada protein, but both exhibit normal DNA phosphotriester repair. These same proteins also exhibit delayed and sluggish induction of the ada and alkA genes. Since the C-terminal 06-methylguanine methyltransferase domain of Ada is not implicated in the direct binding of specific DNA sequences, this part of the Ada protein is likely to play an alternative mechanistic role in gene activation, either by promoting Ada dimerization, or via direct contacts with RNA polymerase.
Proceedings of the National Academy of Sciences, 1985
The inducible resistance to alkylation mutagenesis and killing in Escherichia coli (the adaptive response) is controlled by the ada gene. The Ada protein acts both as a positive regulator of the response and as a DNA repair enzyme, correcting premutagenic O6-alkylguanine in DNA by suicidal transfer of the alkyl group to one of its own cysteine residues. We have determined the DNA sequence of the cloned ada+ gene and its regulatory region. The data reveal potential sites of ada autoregulation. Amino acid sequence determinations show that the active center for the O6-methylguanine-DNA methyltransferase is located close to the polypeptide COOH terminus and has the unusual sequence-Pro-Cys-His-, preceded by a very hydrophobic region. These same structural features are present at the active site of thymidylate synthase, suggesting a common chemical mechanism for activation of the cysteine.
Journal of Bacteriology, 2000
Alkylation damage to DNA occurs when cells encounter alkylating agents in the environment or when cellular metabolism produces active alkylators. To cope with DNA alkylation, cells have evolved genes that encode proteins with alkylationspecific DNA repair activities. In Escherichia coli, the main response specific for alkylation damage has been called the adaptive response (53). The adaptive response genes are induced upon exposure to exogenous alkylators by Ada-dependent induction, and also during stationary phase by rpoS-dependent gene expression, possibly to prevent accumulation of DNA damage due to increased endogenous production of alkylating agents. Recent studies of the regulatory mechanisms of Ada protein and the various responses of the individual promoters regulated by this protein has revealed a complexity of regulation not initially recognized. In this review we describe the roles of the Ada-regulated genes and the regulatory mechanisms that activate gene expression from the three Ada-dependent promoters. We will focus on Ada-dependent induction of the adaptive response genes, fine tuning of individual gene expression according to the growth phase, and the role played by Ada in shutting off the adaptive response. Ada-DEPENDENT REGULATION OF THE ADAPTIVE RESPONSE GENES
PLoS ONE, 2013
Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada) response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i) the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii) the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB), characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB), PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems against alkylating agents of exo-and endogenous origin. Figure 2. The multiple protein alignment of E. coli K-12 DH10B EcAlkA (locus tag: ECDH10B_2218), P. putida KT2440 PpAlkA (PP_0705), P. putida GB-1 PpAlkA2 (PputGB1_2545), and D. radiodurans DrAlkA (DR_2584) generated with ClustalW (A).