Excision of Oxidative Cytosine Modifications from γ-Irradiated DNA byEscherichia coliEndonuclease III and Human Whole-Cell Extracts

Wagner, J. Richard; Blount, Benjamin C.; Weinfeld, Michael

doi:10.1006/abio.1996.0010

Cited by 39 publications

(32 citation statements)

References 7 publications

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“…Although the relative rates of excision of several products have been compiled for Endo III and various DNA substrates, they have failed to distinguish between the excision of cytosine glycols and 5-hydroxycytosine (23,45). In the present study, the excision of cytosine glycols and 5-hydroxycytosine was determined by comparison of the rates of excision from freshly oxidized and heated polymers.…”

Section: Discussionmentioning

confidence: 99%

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Dehydration, deamination and enzymatic repair of cytosine glycols from oxidized poly(dG-dC) and poly(dI-dC)

Tremblay

Wagner

2007

Nucleic Acids Research

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Cytosine glycols (5,6-dihydroxy-5,6-dihydrocytosine) are initial products of cytosine oxidation. Because these products are not stable, virtually all biological studies have focused on the stable oxidation products of cytosine, including 5-hydroxycytosine, uracil glycols and 5-hydroxyuracil. Previously, we reported that the lifetime of cytosine glycols was greatly enhanced in double-stranded DNA, thus implicating these products in DNA repair and mutagenesis. In the present work, cytosine and uracil glycols were generated in double-stranded alternating co-polymers by oxidation with KMnO4. The half-life of cytosine glycols in poly(dG-dC) was 6.5 h giving a ratio of dehydration to deamination of 5:1. At high substrate concentrations, the excision of cytosine glycols from poly(dG-dC) by purified endonuclease III was comparable to that of uracil glycols, whereas the excision of these substrates was 5-fold greater than that of 5-hydroxycytosine. Kinetic studies revealed that the Vmax was several fold higher for the excision of cytosine glycols compared to 5-hydroxycytosine. In contrast to cytosine glycols, uracil glycols did not undergo detectable dehydration to 5-hydroxyuracil. Replacing poly(dG-dC) for poly(dI-dC) gave similar results with respect to the lifetime and excision of cytosine glycols. This work demonstrates the formation of cytosine glycols in DNA and their removal by base excision repair.

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Section: Discussionmentioning

confidence: 99%

“…Authentic standards of 5-hydroxycytosine and 5-hydroxyuracil were prepared by an established method (22). Stable isotopes (+3 amu) of cis and trans uracil glycols, 5-hydroxycytosine, 5-hydroxyuracil were prepared from 15 N 2 13 C-labeled urea (Cambridge Isotopes), as previously described (23,24). …”

Section: Methodsmentioning

confidence: 99%

Dehydration, deamination and enzymatic repair of cytosine glycols from oxidized poly(dG-dC) and poly(dI-dC)

Tremblay

Wagner

2007

Nucleic Acids Research

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“…Endonuclease III (Endo III) was the first glycosylase discovered to contain this metal cofactor [8]. Endo III removes a wide variety of oxidized pyrimidines from DNA and contains the helixhairpin-helix (HhH) recognition motif [11][12][13][14][15][16][17][18][19]. MutY, structurally similar to Endo III [18][19][20][21], is another BER glycosylase that contains a [4Fe-4S] cluster [20].…”

Section: Introductionmentioning

confidence: 99%

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

Boal

Yavin

Barton

2007

Journal of Inorganic Biochemistry

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The [4Fe-4S] cluster is ubiquitous to a class of base excision repair enzymes, in organisms ranging from bacteria to man, and was first considered as a structural element, owing to its redox stability under physiological conditions. When studied bound to DNA, two of these repair proteins (MutY and Endonuclease III from Escherichia coli) display DNA-dependent reversible electron transfer with characteristics typical of high potential iron proteins. These results have inspired a reexamination of the role of the [4Fe-4S] cluster in this class of enzymes. Might the [4Fe-4S] cluster be used as a redox cofactor to search for damaged sites using DNA-mediated charge transport, a process well known to be highly sensitive to lesions and mismatched bases? Described here are experiments demonstrating the utility of DNA-mediated charge transport in characterizing these DNA-binding metalloproteins, as well as efforts to elucidate this new function for DNA as an electronic signaling medium among the proteins.

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“…Two examples, highly homologous (8), are MutY (1) and endonuclease III (Endo III) (2,9), base excision repair enzymes from Escherichia coli (10). MutY, containing 350 residues, acts as a glycosylase to remove adenine from G:A (11-13) and 7,8-dihydro-8-oxo-2-deoxyguanonsine:A mismatches (14-21); Endo III removes pyrimidines damaged by ring saturation, contraction, or fragmentation (22)(23)(24)(25)(26)(27)(28)(29). Although MutY and Endo III have dramatically different substrate recognition features, they both contain a [4Fe4S] 2ϩ cluster (1, 2, 9) within a Cys-X 6 -Cys-X 2 -Cys-X 5 -Cys loop located near the protein C terminus (1,9,30).…”

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confidence: 99%

DNA-mediated charge transport for DNA repair

Boon

Livingston

Chmiel

et al. 2003

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

222

223

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MutY, like many DNA base excision repair enzymes, contains a [4Fe4S] 2؉ cluster of undetermined function. Electrochemical studies of MutY bound to a DNA-modified gold electrode demonstrate that the [4Fe4S] cluster of MutY can be accessed in a DNA-mediated redox reaction. Although not detectable without DNA, the redox potential of DNA-bound MutY is Ϸ275 mV versus NHE, which is characteristic of HiPiP iron proteins. Binding to DNA is thus associated with a change in [4Fe4S] 3؉/2؉ potential, activating the cluster toward oxidation. Given that DNA charge transport chemistry is exquisitely sensitive to perturbations in base pair structure, such as mismatches, we propose that this redox process of MutY bound to DNA exploits DNA charge transport and provides a DNA signaling mechanism to scan for mismatches and lesions in vivo.D NA repair proteins that contain a FeS redox cofactor are ubiquitous (1-7), yet a role for these factors has been lacking. Two examples, highly homologous (8), are MutY (1) and endonuclease III (Endo III) (2, 9), base excision repair enzymes from Escherichia coli (10). MutY, containing 350 residues, acts as a glycosylase to remove adenine from G:A (11-13) and 7,8-dihydro-8-oxo-2-deoxyguanonsine:A mismatches (14-21); Endo III removes pyrimidines damaged by ring saturation, contraction, or fragmentation (22-29). Although MutY and Endo III have dramatically different substrate recognition features, they both contain a [4Fe4S] 2ϩ cluster (1, 2, 9) within a Cys-X 6 -Cys-X 2 -Cys-X 5 -Cys loop located near the protein C terminus (1, 9, 30). Based on sequence alignment, a loop defined by the first two ligating cysteines, called the FCL, is proposed to be a common element of DNA repair proteins (30), present throughout phylogeny (31-37). The function, if any, for these clusters remains undetermined, although the FCL has been proposed as a structural element, aiding in DNA binding (9,30,38). Interestingly, however, MutY is capable of folding without the cluster; the [4Fe4S] 2ϩ cluster adds no stability to the enzyme, but it is critical for substrate binding and catalysis (39). The solvent-accessible [4Fe4S] 2ϩ cluster of Endo III undergoes decomposition with ferricyanide and is resistant to reduction, with an estimated midpoint potential of ϽϪ600 mV for the [4Fe4S] 2ϩ/1ϩ couple (2, 38).Here, we consider whether the [4Fe4S] 2ϩ cluster in MutY can function in DNA-mediated charge transport (CT). Many laboratories have probed DNA-mediated CT chemistry (40-42). Using biochemical, spectroscopic, and electrochemical methods, we have shown that CT through DNA can proceed over long molecular distances in a reaction that is remarkably sensitive to intervening dynamical base pair structure (43-50). DNAmediated CT has been shown to yield oxidative DNA damage from a distance within nucleosome core particles (47) and HeLa cell nuclei (51). DNA binding proteins and peptides have also been shown to modulate and participate in long-range CT chemistry (48, 49), raising the question of the physiological relevance of DNA CT....

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Excision of Oxidative Cytosine Modifications from γ-Irradiated DNA byEscherichia coliEndonuclease III and Human Whole-Cell Extracts

Cited by 39 publications

References 7 publications

Dehydration, deamination and enzymatic repair of cytosine glycols from oxidized poly(dG-dC) and poly(dI-dC)

Dehydration, deamination and enzymatic repair of cytosine glycols from oxidized poly(dG-dC) and poly(dI-dC)

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

DNA-mediated charge transport for DNA repair

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