Thymine methyls and DNA–protein interactions

Ivarie, Robert

doi:10.1093/nar/15.23.9975

Cited by 51 publications

(45 citation statements)

References 38 publications

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“…We recently have shown that replacement of the hydrophobic 5-methyl group of thymine with the bulkier, hydrophilic 5-hydroxymethyl group, results in a significant decrease in binding of the ubiquitous transcription factor, AP-1 (15). Indeed, there are many sequence-specific DNA binding proteins that recognize the 5-methyl group of thymine in the major groove of DNA as a docking site (16), suggesting that HmU accumulation in DNA has great potential to disrupt gene regulation, and thus could be a main reason that multiple overlapping glycosylases exist to repair this lesion out of the DNA of human cells.…”

Section: Introductionmentioning

confidence: 99%

Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

Rogstad

Darwanto

Herring

et al. 2007

Chem. Res. Toxicol.

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The DNA of all organisms is constantly damaged by oxidation. Among the array of damage products is 5-hydroxymethyluracil, derived from oxidation of the thymine methyl group. Previous studies have established that HmU can be a sensitive and valuable marker of DNA damage. More recently, the corresponding deoxynucleoside, 5-hydroxymethyl-2′-deoxyuridine (HmdU) has proven to be valuable for the introduction of controlled amounts of a single type of damage lesion into the DNA of replicating cells, which is subsequently repaired by the base excision repair pathway. Complicating the study of HmU formation and repair, however, is the known chemical reactivity of the hydroxymethyl group of HmU under conditions used to hydrolyze DNA. In the work reported here, this chemical property has been exploited by creating conditions that convert HmU to the corresponding methoxymethyluracil (MmU) derivative that can be further derivatized to the 3,5-bis-(trifluoromethyl)benzyl analog. This derivatized compound can be detected by gas chromatographynegative chemical ionization-mass spectrometry (GC-NCI-MS) with good sensitivity. Using isotopically enriched exogenous HmdU and human osteosarcoma cells (U2OS) in culture, we demonstrate that this method allows for the measurement of HmU in DNA formed from incorporation of exogenous HmdU. We further demonstrate that addition of isotopically enriched uridine to the culture medium allows for the simultaneous measurement of DNA replication and repair kinetics. This sensitive and facile method should prove valuable for studies on DNA oxidation damage and repair in living cells.

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Section: Introductionmentioning

confidence: 99%

Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

Rogstad

Darwanto

Herring

et al. 2007

Chem. Res. Toxicol.

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“…Methods for detecting contacts between thymines and proteins have been described (7,8). They involve either expensive synthesis of oligonucleotides and the use of base analogues that yield precise and reliable information about the importance of methyl groups of thymines (7) or hydrazine treatment of DNA that results in a complete removal of the thymine base and opening of the sugar ring (8).…”

Section: Introductionmentioning

confidence: 99%

“…In addition, there are thymine residues conserved in both subgroups. In an attempt to study directly the participation of thymine residues within the HRE in specific binding of the hormone receptors, and in the discrimination between different HREs, we have searched for appropriate chemical procedures.Methods for detecting contacts between thymines and proteins have been described (7,8). They involve either expensive synthesis of oligonucleotides and the use of base analogues that yield precise and reliable information about the importance of methyl groups of thymines (7) or hydrazine treatment of DNA that results in a complete removal of the thymine base and opening of the sugar ring (8).…”

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confidence: 99%

Contacts between steroid hormone receptors and thymines in DNA: an interference method.

Truss¹,

Chalepakis²,

Beato³

1990

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

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Understanding the mechanisms by which regulatory proteins recognize genetic information stored in DNA relies on the availability of methods to analyze their interaction with individual nucleotides and their reactive groups. Here we describe the use of KMnO4 to analyze the contacts between steroid hormone receptors and thymines within a hormone responsive element. Although several pyrimidine residues are highly conserved among different receptor binding sites their participation in sequence recognition has not been directly studied. Using an interference procedure based on selective modification of the thymine ring by KMnO4, we detect intimate contacts between the glucocorticoid or progesterone receptors and three thymine residues within the promoter distal receptor binding site of the mouse mammary tumor virus (-190/-160). A comparison of binding data obtained with oligonucleotides containing desoxyuridine, bromodesoxyuridine, cytosine, or 5'-methylcytosine instead of thymines demonstrates that the methyl group of those three thymines contributes to the free energy of binding. This simple method could be of general utility for the study of sequence-specific protein-DNA interactions.Regulation of gene expression at the transcriptional level is accomplished primarily through specific interaction of regulatory proteins with defined DNA sequence elements (1). During the past few years, our understanding of the mechanism responsible for specific binding of proteins to DNA has made considerable progress due to the availability of highresolution analytical methods. In addition to classical footprinting methods (2) that serve to determine the limits of the DNA sequences recognized by particular proteins, chemical modification procedures have been useful for identifying contacts between the proteins and individual reactive groups of the phosphate-sugar backbone (3, 4) or the nucleotide bases. Among the latter, protection against, or interference by, methylation of purines with dimethyl sufate has been widely used (5). Inspection of the DNA double helix through its major groove shows that the 5'-methyl group of thymine is one of the salient features that distinguishes different base pairs. To further the understanding of the interaction between regulatory proteins, in particular steroid hormone receptors, and DNA, we have developed a useful procedure for analyzing the role of thymine residues in specific protein-DNA recognition.Steroid hormones modulate gene expression by virtue of their binding to specific receptor proteins that are members of the superfamily of nuclear receptors (6). These proteins interact with a specific class of DNA sequences located in the vicinity of the regulated promoters called hormone responsive elements (HREs).A comparison of the different HREs reported so far allows classification into two subgroups: (i) the GRE subgroup, with the structure GGTACAnnnTGTYCT, that mediates response to glucocorticoids, progestins, androgens, and mineralocorticoids; and (ii) the ERE subgroup, with the str...

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“…Methyl groups were introduced at the 5 position of cytosine with the C-C bond length constrained to that previously determined in crystalline Z-DNA (23). As with thymine methyls, cytosine methyls are not expected to perturb B-DNA conformation, since both pyrimidine methyls occupy roughly the same position in the major groove of the B-DNA helix (24,25). This expectation was consistent with the molecular modeling carried out here, in which the energy-minimized structure was found to be roughly isomorphic to the B-DNA conformation adopted by the structure prior to minimization.…”

mentioning

confidence: 99%

Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation.

Smith

Kaplan

Sowers

et al. 1992

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

138

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The properties of the methyl-iected DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) suggest that it is the enzyme that ntains patterns of methylation in the human genome. Proposals for the enzyme's mhanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate. We have used an oligodeoxynuceotide containg 5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine,, suggesting that it comprises an enzymelinked 5-substituted dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transi mutation are epially prevalent at CG sites in human DNA, we have used high-performance liquid chromatography to searchfor thymidine that might be generated by hydrolysis during the methyl triaser reaction. Despite the potential for deamination ient in the formation of the intermedite, the methylferae did not produce detectable amounts of thymidine. The data suggest that the alit of the human methylter to preserve genetic information when copying a methylation pattern (i.e., its fdelity) is comparable to the ability of a amm DNA polymerase to preserve genetic information when copying a DNA sequence.Thus the high frequency of C-to-T transitions at CG sites in human DNA does not appear to be due to the normal enzymatic maintenance of methylation patterns.Proposals for the mechanism of action of DNA methyltransferases (6) suggest production ofa dihydrocytosine intermediate (Fig. 2). These derivatives are extremely susceptible to hydrolytic deamination, since saturation ofthe 5-6 bond increases the PKa of the ring significantly (7). This increase will facilitate protonation ofdihydrocytosine and thereby increase the rate of subsequent hydrolytic deamination in a form of the additionelimination mechanism (8, 9) depicted in Fig. 2A. Although the expected enzyme-DNA intermediate has been detected with a prokaryotic methyltransferase (10), prior to the experiments described here, direct evidence for its formation by a eukaryotic methyltransferase had not, to our knowledge, been reported. Even so, we have pointed out that the production of this nonplanar intermediate in DNA would be consistent with the observed preference of the human enzyme for unusual DNA structures (11).Since the putative intermediate (Fig. 2) might also be susceptible to deamination during catalysis (12), the enzymatic maintenance of methylation patterns could contribute to the high frequency of C-to-T transitions at the CG dinucleotide pair that are thought to be the result of deamination of methylcytosine to thymidine (13-15). Since the C-to-T mutation frequency observed in vivo at 5-methylcytosine relative to that a...

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Thymine methyls and DNA–protein interactions

Cited by 51 publications

References 38 publications

Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

Contacts between steroid hormone receptors and thymines in DNA: an interference method.

Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation.

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