HPLC product analysis demonstrated that Iz is a key oxidation product of G through 8-oxoG in DNA photosensitized with riboflavin or anthraquinone. Photoreaction of AQ-linked oligomer confirmed that Iz is formed from 8-oxoG through long-range hole migration. Two sets of primer extension experiments demonstrated that Iz can specifically pair with G in vitro. Specific Iz-G base pair formation can explain the G-C-->C-G transversion mutations that appear under oxidative conditions.

Considerable interest has recently arisen in the one-electron oxidations of DNA in connection with DNA damage caused by ionizing radiation, oxidizing agents, two-photon photoionization by a high-intensity laser pulse, and photoirradiation with photosensitizers. 1 We and others have found a common feature in that one-electron oxidation of DNA selectively generates piperidinesensitive alkaline-labile sites at the 5′-guanine (G) of 5′-GG-3′ sequences. 2,3 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxoG) has been repeatedly claimed to be the major oxidation product ultimately leading to the piperidine-dependent G cleavage during the one-electron photooxidation of duplex DNA in the presence of various electron-accepting photosensitizers. 4 Recently, Cullis et al. have demonstrated that the 8-oxoG-containing site is not efficiently cleaved by hot piperidine treatment (90°C, 20 min). 5 While the alkaline-labile sites have been suggested to be 2,2diaminooxazolone (Z) and/or 2-aminoimidazolone (Iz) containing sites, 1f,5 which were already identified as the oxidation products of deoxyguanosine by Cadet et al., 6 the direct evidence for the formation of these sites in double-strand DNA has not been documented.To elucidate the real structure of the alkaline-labile site derived from the guanine cation radical, we have carried out the detailed product analysis of photoirradiated 5′-d(TTGGTA)-3′ and calf thymus DNA in the presence of riboflavin. We found that the Iz-containing oligomer is produced as a major isolatable product and the 8-oxoG-containing oligomer is only a minor product. Figure 1a shows the reverse phase HPLC profile of photoirradiated single-stranded 5′-d(TTGGTA)-3′ in the presence of riboflavin, showing the formation of two major peaks (peaks 1 and 2) eluted at 35.1 and 37.9 min, respectively. 7 Prolonged low temperature digestion (0°C, 15 h) of both products provided exactly the same HPLC profile showing the formation of dG, dT, dA, and Iz in a ratio of 1:1:3:1. 8 Electrospray mass spectra (1783, ESMS) confirmed that one G of 5′-d(TTGGTA)-3′ is oxidized to Iz in both products. Hot piperidine treatment (1 M, 90°C, 20 min) of peak 1 gave TTGp and pTA, whereas the same treatment of peak 2 gave TTp and pGTA, indicating that the structures of peaks 1 and 2 were 5′-d(TTGIzGTA)-3′ (1) and 5′d(TTIzGTA)-3′ (2), respectively. A similar procedure for the isolated peak 3 demonstrated that its structure is 5′-d(TTIzIzTA)-3′ (3). ESMS indicated that both peaks 4 and 5 have the same molecular weight of 1826. These were also observed in the photoirradiation of the 8-oxoG-containing hexamers 5′-d(TT 8OXO -GGTA)-3′ and 5′-d(TTG 8OXO GTA)-3′ in the presence of riboflavin. However, further characterization of these products has not been successful due to their thermal instability. Figure 1b shows the HPLC profile of the riboflavin-sensitized photooxidation of 5′-d(TTGGTA)-3′ in the presence of the complementary oligomer 5′-d(ATACCAAA)-3′. 9 The results of the quantitative product analysis are summarized in Table 1. It(1) (a) For ...

The Ewing’s sarcoma (EWS) oncogene contains an N‐terminal transcription activation domain and a C‐terminal RNA‐binding domain. Although the EWS activation domain is a potent transactivation domain that is required for the oncogenic activity of several EWS fusion proteins, the normal role of intact EWS is poorly characterized because little is known about its nucleic acid recognition specificity. Here we show that the Arg‐Gly‐Gly (RGG) domain of the C‐terminal in EWS binds to the G‐rich single‐stranded DNA and RNA fold in the G‐quadruplex structure. Furthermore, inhibition of DNA polymerase on a template containing a human telomere sequence in the presence of RGG occurs in an RGG concentration‐dependent manner by the formation of a stabilized G‐quadruplex DNA–RGG complex. In addition, mutated RGG containing Lys residues replacing Arg residues at specific Arg‐Gly‐Gly sites and RGG containing Arg methylated by protein arginine N‐methyltransferase 3 decrease the binding ability of EWS to G‐quadruplex DNA and RNA. These findings suggest that the RGG of EWS binds to G‐quadruplex DNA and RNA via the Arg residues in it.

New mechanism for G:C–C:G transversions: Here, we show for the first time that guanine is preferentially incorporated opposite 2,2,4‐triamino‐5(2 H)‐oxazolone (Oz) by eukaryotic DNA polymerases α, β, and ε. The proposed Oz:G base pair is presented.

Guanine is the most readily oxidized of the four DNA bases, and guanine oxidation products cause G:C-T:A and G:C-C:G transversions through DNA replication. 8-Oxo-7,8-dihydroguanine (8-oxoG) causes G:C-T:A transversions but not G:C-C:G transversions, and is more readily oxidized than guanine. This review covers four major findings. (i) 2,2,4-Triamino-5(2H)-oxazolone (Oz) is produced from guanine and 8-oxoG under various oxidative conditions. Guanine is incorporated opposite Oz by DNA polymerases, except REV1. (ii) Several enzymes exhibit incision activity towards Oz. (iii) Since the redox potential of GG is lower than that of G, contiguous GG sequences are more readily oxidized by a one-electron oxidant than a single guanine, and OzOz is produced from GG in double-stranded DNA. Unlike most DNA polymerases, DNA polymerase ζ efficiently extends the primer up to full-length across OzOz. (iv) In quadruplex DNA, 3′-guanine is mainly damaged by one-electron oxidation in quadruplex DNA, and this damage depends on the highest occupied molecular orbital (HOMO). The oxidation products in quadruplex DNA are different from those in single-stranded or double-stranded DNA.