Ada regulon

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In DNA repair, the Ada regulon is a set of genes whose expression is essential to adaptive response (also known as "Ada response", hence the name), which is triggered in prokaryotic cells by exposure to sub-lethal doses of alkylating agents. This allows the cells to tolerate the effects of such agents, which are otherwise toxic and mutagenic.

Contents

The Ada response includes the expression of four genes: ada, alkA, alkB, and aidB. The product of ada gene, the Ada protein, is an activator of transcription of all four genes. DNA bases damaged by alkylation are removed by distinct strategies.

Alkylating agents

The alkylating agents from a group of mutagens and carcinogens that modify DNA by alkylation. Alkyl base lesions can arrest replication, interrupt transcription, or signal the activation of cell cycle checkpoints or apoptosis. In mammals, they could be involved in carcinogenesis, neurodegenerative disease and aging. The alkylating agents can introduce methyl or ethyl groups at all of the available nitrogen and oxygen atoms in DNA bases, providing a number of lesions.

The majority of evidence indicates that among the 11 identified base modification two, 3-methyladenine (3meA) and O6-methylguanine (O6-meG), are mainly responsible for the biological effects of alkylation agents. [1]

Roles of ada-regulated genes

The Ada protein is composed of two major domains, a C-terminal domain and an N-terminal one, linked by a hinge region susceptible to proteolytic cleavage. These domains can function independently. AdaCTD transfers methyl adducts from O6-meG and O4-meG onto its Cys-321 residue, whereas AdaNTD demethylates methyl-phosphotriesters by methyl transfer onto its Cys-38 residue. [2] [3] [4]

The alkA gene encodes a glycosylase that repairs a variety of lesions including N-7-Methylguanine and N-3-Methylpurines and O2-methyl pyrimidines. [2] The AlkA protein removes a damaged base from the sugar-phosphate backbone by cleaving the glycosylic bond attaching the base to the sugar, producing an abasic site. Further processing of the abasic site by AP endonucleases, polymerase I, and ligase then completes the repair. [5]

AlkB, one of the Escherichia coli adaptive response proteins, uses an α ketoglutarate/Fe(II)-dependent mechanism that, by chemical oxidation, removes a variety of alkyl lesions from DNA, thus affording protection of the genome against alkylation. [6]

The AidB protein has been supposed to take part in the degradation of endogenous alkylating agents. [7] [8] It shows some homology to acyl-CoA oxidases and those containing flavins. [7] Recent observations suggest that AidB may bind to double-stranded DNA and take part in its dealkylation. [8] However, to determine the precise function of AidB further investigations are necessary.

Regulation of transcription

Ada Regulatory network Adaptive Response (Ada) Regulatory Network.png
Ada Regulatory network

The Ada response includes the expression of four genes: ada, alkA, alkB, and aidB. The product of the ada gene, the Ada protein is an activator of transcription of all four genes.

Ada has two active methyl acceptor cysteine residues that are required for demethylation of DNA. Both sites can become methylated when Ada protein transfers the methyl group from the appropriate DNA lesions to itself. This reaction is irreversible and methylated Ada (me-Ada) can act as a transcriptional activator.

The Ada protein activates the transcription of the Ada regulon in two different ways. In case of the ada-alkB operon, and the aidB promoter, the N-terminal domain (AdaNTD) is involved in DNA binding and interacts with the a unit of RNA polymerase, whereas and the methylated C-terminal domain (me-AdaCTD) interacts with the σ70 subunit of RNA polymerase. Although these interactions are independent, both are necessary for transcription activation.

For activation of alkA gene, the AdaNTD interacts with both, the α and σ subunits of RNA polymerase, and activates transcription. In contrast to the ada and aidB promoters, the unmethylated form of the Ada protein, as well as methylated form of the AdaNTD, is able to activate the transcription at alkA.

Methylated Ada is able to activate transcription by σS as well as σ70 at both the ada and aidB promoters. [9] [10] In contrast, not only does me-Ada fail to stimulate alkA transcription by σS, but it negatively affects σS dependent transcription.

Intracellular concentrations of σS increase when the cells reach stationary phase; this in turn results in a me-Ada mediated decrease in the expression of AlkA. Therefore, an increase in expression of the adaptive response genes, in parallel with the expression of genes producing endogenous alkylators during the stationary phase, prevents alkylation damage to DNA and mutagenesis.

Homologues of the Ada regulon in humans

In human cells, the alkyltransferase activity is the product of the MGMT gene. [11] [12] The 21.7 kDa MGMT protein is built of amino-acid sequences very similar to those of E. coli alkyltransferases, like Ada. In contrast to the bacterial enzymes it mainly repairs O6meG, whereas removal of the alkyl adduct from O4meT is much slower and significantly less effective. [13] [14] The preferential repair of O6meG is profitable for eukaryotic cells since in experimental animals treated with alkylating carcinogens this lesion is involved in tumor stimulation.

Unlike the Ada and the human MGMT methyltransferases, AlkB and its human homologs hABH2 and hABH3 not only reverse alkylation base damage directly, but they do so catalytically and with a substrate specificity aimed at the base-pairing interface of the G:C and A:T base pairs. [15] [16] [17] Crystal structures of AlkB and its human homologue hABH3 have shown similar overall folds, highlighting conserved functional domains. [18]

Related Research Articles

In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These terms are commonly used in chemistry, biochemistry, soil science, and the biological sciences.

In molecular biology and genetics, transcriptional regulation is the means by which a cell regulates the conversion of DNA to RNA (transcription), thereby orchestrating gene activity. A single gene can be regulated in a range of ways, from altering the number of copies of RNA that are transcribed, to the temporal control of when the gene is transcribed. This control allows the cell or organism to respond to a variety of intra- and extracellular signals and thus mount a response. Some examples of this include producing the mRNA that encode enzymes to adapt to a change in a food source, producing the gene products involved in cell cycle specific activities, and producing the gene products responsible for cellular differentiation in multicellular eukaryotes, as studied in evolutionary developmental biology.

<span class="mw-page-title-main">DNA repair</span> Cellular mechanism

DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA damage, resulting in tens of thousands of individual molecular lesions per cell per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. When normal repair processes fail, and when cellular apoptosis does not occur, irreparable DNA damage may occur, including double-strand breaks and DNA crosslinkages. This can eventually lead to malignant tumors, or cancer as per the two hit hypothesis.

<span class="mw-page-title-main">Methyltransferase</span> Group of methylating enzymes

Methyltransferases are a large group of enzymes that all methylate their substrates but can be split into several subclasses based on their structural features. The most common class of methyltransferases is class I, all of which contain a Rossmann fold for binding S-Adenosyl methionine (SAM). Class II methyltransferases contain a SET domain, which are exemplified by SET domain histone methyltransferases, and class III methyltransferases, which are membrane associated. Methyltransferases can also be grouped as different types utilizing different substrates in methyl transfer reactions. These types include protein methyltransferases, DNA/RNA methyltransferases, natural product methyltransferases, and non-SAM dependent methyltransferases. SAM is the classical methyl donor for methyltransferases, however, examples of other methyl donors are seen in nature. The general mechanism for methyl transfer is a SN2-like nucleophilic attack where the methionine sulfur serves as the leaving group and the methyl group attached to it acts as the electrophile that transfers the methyl group to the enzyme substrate. SAM is converted to S-Adenosyl homocysteine (SAH) during this process. The breaking of the SAM-methyl bond and the formation of the substrate-methyl bond happen nearly simultaneously. These enzymatic reactions are found in many pathways and are implicated in genetic diseases, cancer, and metabolic diseases. Another type of methyl transfer is the radical S-Adenosyl methionine (SAM) which is the methylation of unactivated carbon atoms in primary metabolites, proteins, lipids, and RNA.

<span class="mw-page-title-main">DNA adenine methylase</span> Prokaryotic enzyme

DNA adenine methylase, (Dam methylase) (also site-specific DNA-methyltransferase (adenine-specific), EC 2.1.1.72, modification methylase, restriction-modification system) is an enzyme that adds a methyl group to the adenine of the sequence 5'-GATC-3' in newly synthesized DNA. Immediately after DNA synthesis, the daughter strand remains unmethylated for a short time. It is an orphan methyltransferase that is not part of a restriction-modification system and regulates gene expression. This enzyme catalyses the following chemical reaction

The adaptive response is a form of direct DNA repair in E. coli that protects DNA from damage by external agents or by errors during replication. It is initiated against alkylation, particularly methylation, of guanine or thymine nucleotides or phosphate groups on the sugar-phosphate backbone of DNA. Under sustained exposure to low-level treatment with alkylating mutagens, E. coli can adapt to the presence of the mutagen, rendering subsequent treatment with high doses of the same agent less effective.

<span class="mw-page-title-main">Crosslinking of DNA</span> Phenomenon in genetics

In genetics, crosslinking of DNA occurs when various exogenous or endogenous agents react with two nucleotides of DNA, forming a covalent linkage between them. This crosslink can occur within the same strand (intrastrand) or between opposite strands of double-stranded DNA (interstrand). These adducts interfere with cellular metabolism, such as DNA replication and transcription, triggering cell death. These crosslinks can, however, be repaired through excision or recombination pathways.

Ada, also called as O6 alkyl guanine transferase I (O6 AGT I), is an enzyme induced by treatment of bacterial cells with alkylating agents that mainly cause methylation damage. This phenomenon is called the adaptive response hence the name. Ada transfers the alkyl group from DNA bases and sugar-phosphate backbone to a cysteine residue, inactivating itself. Consequently, it reacts stoichiometrically with its substrate rather than catalytically and is referred to as a suicide enzyme. Methylation of Ada protein converts it into a self transcriptional activator, inducing its own gene expression and the expression of other genes which together with Ada help the cells repair alkylation damage. Ada removes the alkyl group attached to DNA bases like guanine (O6-alkyl guanine) or thymine (O4-alkyl thymine) and to the oxygen of the phosphodiester backbone of the DNA. However, Ada shows greater preference for O6- alkyl guanine compared to either O4-thymine and alkylated phosphotriesters. Ada enzyme has two active sites, one for the alkylated guanines and thymines and the other for alkylated phosphotriesters.

AlkB (Alkylation B) is a protein found in E. coli, induced during an adaptive response and involved in the direct reversal of alkylation damage. AlkB specifically removes alkylation damage to single stranded (SS) DNA caused by SN2 type of chemical agents. It efficiently removes methyl groups from 1-methyl adenines, 3-methyl cytosines in SS DNA. AlkB is an alpha-ketoglutarate-dependent hydroxylase, a superfamily non-haem iron-containing proteins. It oxidatively demethylates the DNA substrate. Demethylation by AlkB is accompanied with release of CO2, succinate, and formaldehyde.

O6-alkylguanine DNA alkyltransferase II previously known as O6 Guanine transferase (ogt) is a bacterial protein that is involved in DNA repair together with Ada.

In enzymology, a methylated-DNA-[protein]-cysteine S-methyltransferase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">O-6-methylguanine-DNA methyltransferase</span> Mammalian protein found in Homo sapiens

O6-alkylguanine DNA alkyltransferase (also known as AGT, MGMT or AGAT) is a protein that in humans is encoded by the O6-methylguanine DNA methyltransferase (MGMT) gene. O6-methylguanine DNA methyltransferase is crucial for genome stability. It repairs the naturally occurring mutagenic DNA lesion O6-methylguanine back to guanine and prevents mismatch and errors during DNA replication and transcription. Accordingly, loss of MGMT increases the carcinogenic risk in mice after exposure to alkylating agents. The two bacterial isozymes are Ada and Ogt.

<span class="mw-page-title-main">DNA-3-methyladenine glycosylase</span> Protein-coding gene in the species Homo sapiens

DNA-3-methyladenine glycosylase also known as 3-alkyladenine DNA glycosylase (AAG) or N-methylpurine DNA glycosylase (MPG) is an enzyme that in humans is encoded by the MPG gene.

6-<i>O</i>-Methylguanine Chemical compound

6-O-Methylguanine is a derivative of the nucleobase guanine in which a methyl group is attached to the oxygen atom. It base-pairs to thymine rather than cytosine, causing a G:C to A:T transition in DNA.

DNA Polymerase V is a polymerase enzyme involved in DNA repair mechanisms in bacteria, such as Escherichia coli. It is composed of a UmuD' homodimer and a UmuC monomer, forming the UmuD'2C protein complex. It is part of the Y-family of DNA Polymerases, which are capable of performing DNA translesion synthesis (TLS). Translesion polymerases bypass DNA damage lesions during DNA replication - if a lesion is not repaired or bypassed the replication fork can stall and lead to cell death. However, Y polymerases have low sequence fidelity during replication. When the UmuC and UmuD' proteins were initially discovered in E. coli, they were thought to be agents that inhibit faithful DNA replication and caused DNA synthesis to have high mutation rates after exposure to UV-light. The polymerase function of Pol V was not discovered until the late 1990s when UmuC was successfully extracted, consequent experiments unequivocally proved UmuD'2C is a polymerase. This finding lead to the detection of many Pol V orthologs and the discovery of the Y-family of polymerases.

DNA-3-methyladenine glycosylase II is an enzyme that catalyses the following chemical reaction:

DNA-formamidopyrimidine glycosylase is an enzyme with systematic name DNA glycohydrolase . FPG is a base excision repair enzyme which recognizes and removes a wide range of oxidized purines from correspondingly damaged DNA. It was discovered by Zimbabwean scientist Christopher J. Chetsanga in 1975.

Bernd Kaina, born on 7 January 1950 in Drewitz, is a German biologist and toxicologist. His research is devoted to DNA damage and repair, DNA damage response, genotoxic signaling and cell death induced by carcinogenic DNA damaging insults.

<span class="mw-page-title-main">Alkb homolog 3, alpha-ketoglutaratedependent dioxygenase</span> Protein-coding gene in the species Homo sapiens

AlkB homolog 3, alpha-ketoglutaratedependent dioxygenase is a protein that in humans is encoded by the ALKBH3 gene.

Pharmacoepigenetics is an emerging field that studies the underlying epigenetic marking patterns that lead to variation in an individual's response to medical treatment.

References

  1. ↑ Singer B (1976) All oxygens in nucleic acids react with carcinogenic ethylating agents. Nature 264: 333–339
  2. 1 2 Lindahl T, Sedgwick B, Sekiguchi M, Nakabeppu Y (1988) Regulation and expression of the adaptive response to alkylating agents. Annual Review of Biochemistry. 57: 133–157
  3. ↑ Moore MH, Gulbis JM, Dodson EJ, Demple B, Moody PC (1994) Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli. EMBO Journal. 13:1495–1501.
  4. ↑ He C, Wei H, Verdine GL (2003). Converting the sacrificial DNA repair protein N-Ada into a catalytic methyl phosphotriester repair enzyme. Journal of the American Chemical Society. 125: 1450–1451.
  5. ↑ Volkert, M. R. 1988. Adaptive response of Escherichia coli to alkylation damage. Environmental and Molecular Mutagenesis. 11:241–255.
  6. ↑ Deyu Li, James C. Delaney, Charlotte M. Page, et al., "Repair of DNA Alkylation Damage by the Escherichia coli Adaptive Response Protein AlkB as Studied by ESI-TOF Mass Spectrometry," Journal of Nucleic Acids, vol. 2010, Article ID 369434, 9 pages, 2010. doi : 10.4061/2010/369434
  7. 1 2 Landini P, Hajec LI, Volkert MR (1994) Structure and transcriptional regulation of the Escherichia coli adaptive response gene aidB. Journal of Bacteriology. 176: 6583–6589.
  8. 1 2 Rohankhedkar MS, Mulrooney SB, Wedemeyer WJ, Hausinger RP (2006) The AidB component of the Escherichia coli adaptive response to alkylating agents is a flavin containing, DNA-binding protein. Journal of Bacteriology. 188:223–230.
  9. ↑ Landini, P., L. I. Hajec, L. H. Nguyen, R. R. Burgess, and M. R. Volkert.1996. The leucine-responsive protein (lrp) acts as a specific repressor for sigmaS -dependent transcription of the Escherichia coli aidB gene. Molecular Microbiology. 20:947–955.
  10. ↑ Taverna, P., and B. Sedgwick. 1996. Generation of endogenous methylating agents by nitrosation in Escherichia coli. Journal of Bacteriology. 178:5105–5111.
  11. ↑ Harris AL, Karran P, Lindahl T (1983) O6-Methylguanine-DNA methyltransferase of human lymphoid cells: structural and kinetic properties and absence in repair deficient cells. Cancer Res 43: 3247–3252.
  12. ↑ Kataoka H, Hall J, Karran P (1986) Complementation of sensitivity to alkylating agents in Escherichia coli and Chinese hamster ovary cells by expression of a cloned bacterial DNA repair gene. EMBO Journal. 5:3195–3200.
  13. ↑ Brennand J, Margison GP (1986) Expression in mammalian cells of a truncated Escherichia coli gene coding for O6-alkylguanine alkyltransferase reduces the toxic effects of alkylating agents. Carcinogenesis 7: 2081–2084.
  14. ↑ Koike G, Maki H, Takeya H, Hayakawa H, Sekiguchi M (1990) Purification, structure, and biochemical properties of human O6-methylguanine-DNA methyltransferase. Journal of Biological Chemistry. 265:14754–14762.
  15. ↑ Trewick, S. C., Henshaw, T. F., Hausinger, R. P., Lindahl, T. & Sedgwick, B. (2002) Nature 419, 174–178.
  16. ↑ Falnes, P. O., Johansen, R. F. & Seeberg, E. (2002) Nature 419, 178–182.
  17. ↑ Duncan, T., Trewick, S. C., Koivisto, P., Bates, P. A., Lindahl, T. & Sedgwick, B. (2002) Proc. Natl. Acad. Sci. USA 99, 16660–16665.
  18. ↑ Jadwiga N & Elżbieta G (2007), Bacterial DNA repair genes and their eukaryotic homologues: 3. AlkB dioxygenase and Ada methyltransferase in the direct repair of alkylated DNA. Acta Biochemica Polonica, Vol. 54 No. 3/2007, 459–468.
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