Synthesis and template properties of an ethyl phosphotriester modified decadeoxyribonucleotide

Miller, Paul S.; Chandrasegaran, Srinivasan; Dow, Donna L.; Pulford, Stephen M.; Kan, Lou Sing

doi:10.1021/bi00265a014

Cited by 66 publications

(30 citation statements)

References 25 publications

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“…§1734 solely to indicate this fact. G-A-T-T-G-G) in 0.005 M sodium phosphate buffer, pH 7 A-G-C-T-T-G-G) (9) are shown in the histogram above the left part of spectrum A for comparison. It should also be noted that some spikes which are scattered on the spectra were generated by the Bruker WM-500 spectrometer.…”

Section: Resultsmentioning

confidence: 99%

Detection of a guanine X adenine base pair in a decadeoxyribonucleotide by proton magnetic resonance spectroscopy.

Kan

Chandrasegaran

Pulford

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

134

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A decadeoxyribonucleotide, d(C-C-A-A-G-A-T-T-G-G) (I), forms a duplex in solution. The base pairing pattern in this duplex was studied by proton nuclear magnetic resonance spectroscopy. Five NH...N hydrogen-bonded proton resonances were observed, and they were assigned by nuclear Overhauser enhancement experiments as well as by comparison to five previously assigned NH...N hydrogen-bonded proton resonances in a self-complementary duplex of similar sequence, d(C-C-A-A-G-C-T-T-G-G) (II). The results suggest that the central -G-A- residues of I form G X A base pairs in the helical state. The fact that the H2 proton of A at the sixth position from the 5' end of I showed nuclear Overhauser enhancement when the NH...N hydrogen-bonded proton resonance of G X A was irradiated suggests that the bases of the G X A base pair are oriented in an anti-anti conformation. Comparison of the linewidths at the half height of the NH...N hydrogen-bonded proton resonances of I at 1 degree C suggest that the G X A base pairs are less stable than adjacent A X T base pairs.

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Section: Resultsmentioning

confidence: 99%

Detection of a guanine X adenine base pair in a decadeoxyribonucleotide by proton magnetic resonance spectroscopy.

Kan

Chandrasegaran

Pulford

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

134

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“…Cys-69 is required for demethylation of phosphomethyltriesters in the sugarphosphate backbone. This lesion is apparently innocuous, since Ada repairs only one of two stereoisomers (16,36,37,72), leaving the other to remain in DNA with no apparent deleterious consequences (28,40). Although methylated phosphates are innocuous, this lesion is readily produced by methylating agents (37) and provides a sensitive regulatory signal that leads to induction of the Ada regulon.…”

Section: Ada-dependent Regulation Of the Adaptive Response Genesmentioning

confidence: 99%

Regulatory Responses of the Adaptive Response to Alkylation Damage: a Simple Regulon with Complex Regulatory Features

Landini

Volkert

2000

J Bacteriol

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2Alkylation 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 GENESThe adaptive response set of genes is comprised of the ada, alkA, alkB, and aidB genes. Expression of these genes is regulated by Ada, and their induction provides protection against alkylation damage to DNA. The ada gene product has both repair and regulatory activities. These two activities are closely tied to one another, as the Ada protein must be activated to perform its regulatory function and activation is a consequence of its DNA repair activity. Ada has two active methyl acceptor cysteine residues, Cys-69 and Cys-321, that are required for demethylation of DNA. Both sites can become methylated when Ada protein transfers the methyl group from the appropriate substrate DNA lesions to itself. This reaction is irreversible, and methylated Ada ( me Ada) is the terminal end product of the demethylation reaction (31). The two methyl acceptor sites present in Ada differ with respect to the lesions repaired. Cys-321 is the methyl acceptor site required for the removal of methyl groups from either O 6 -methylguanine or O 4 -methylthymine, two highly mutagenic lesions (10, 11). Cys-69 is required for demethylation of phosphomethyltriesters in the sugarphosphate backbone. This lesion is apparently innocuous, since Ada repairs only one of two stereoisomers (16,36,37,72), leaving the other to remain in DNA with no apparent deleterious consequences (28, 40). Although methylated phosphates are innocuous, this lesion is readily produced by methylating agents (37) and provides a sensitive regulatory signal that leads to induction of the Ada regulon. Once Ada protein transfers a methyl group from the methyl-phosphate to the Cys-69 residue, it becomes a transcriptional activator. Thus, the methylated phosphates in DNA serve as the signal that conv...

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“…Sedgwick Methylphosphotriesters in DNA are not known to have a toxic or mutagenic effect on the cells. DNA polymerase I is able to synthesize past these lesions although the rate of replication is reduced (Miller et al 1982). The only known importance of actively repairing methylphosphotriesters in E. coli therefore appears to be the induction of the adaptive response.…”

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confidence: 99%

Molecular Signal for Induction of the Adaptive Response to Alkylation Damage in Esherichia Coli

Sedgwick¹

1987

Journal of Cell Science

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SUMMARYExposure of Escherichia coli to simple alkylating agents, such as methylnitrosourea, causes the induction of at least three DNA repair functions that are under the control of the ada gene. The ada gene product itself repairs several O-methylated lesions in DNA, including methylphosphotriesters and the mutagenic adduct 0 6-methylguanine. The methyl groups are transferred from these lesions on to two different cysteine residues within the Ada protein resulting in selfmethylation. We have found that the Ada protein is converted to an activator of expression of genes involved in the adaptive response after accepting a methyl group from a methylphosphotriester, but not from 0 6-methylguanine. This was shown using the in vitro techniques of DNA-dependent protein synthesis and run-off transcription. Delayed electrophoretic migration and footprinting experiments have shown that the methylated activator of transcription binds to specific DNA sequences immediately upstream from the RNA polymerase binding sites in the promoter regions of the inducible genes. The Ada protein-binding sites contain the common sequence d

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Synthesis and template properties of an ethyl phosphotriester modified decadeoxyribonucleotide

Cited by 66 publications

References 25 publications

Detection of a guanine X adenine base pair in a decadeoxyribonucleotide by proton magnetic resonance spectroscopy.

Detection of a guanine X adenine base pair in a decadeoxyribonucleotide by proton magnetic resonance spectroscopy.

Regulatory Responses of the Adaptive Response to Alkylation Damage: a Simple Regulon with Complex Regulatory Features

Molecular Signal for Induction of the Adaptive Response to Alkylation Damage in Esherichia Coli

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