Generation of 5-(2'-deoxycytidyl)methyl radical and the formation of intrastrand cross-link lesions in oligodeoxyribonucleotides - PubMed (original) (raw)

Qibin Zhang et al. Nucleic Acids Res. 2005.

Abstract

Hydroxyl radical is one of the major reactive oxygen species (ROS) formed from gamma-radiolysis of water or Fenton reaction, and it can abstract one hydrogen atom from the methyl carbon atom of thymine and 5-methylcytosine to give the 5-methyl radical of the pyrimidine bases. The latter radical can also be induced from Type-I photo-oxidation process. Here, we examined the reactivity of the independently generated 5-(2'-deoxycytidyl)methyl radical (I) in single- and double-stranded oligodeoxyribonucleotides (ODNs). It was found that an intrastrand cross-link lesion, in which the methyl carbon atom of 5-methylcytosine and the C8 carbon atom of guanine are covalently bonded, could be formed from the independently generated radical at both GmC and mCG sites, with the yield being much higher at the former site. We also showed by LC-MS/MS that the same cross-link lesions were formed in mC-containing duplex ODNs upon gamma irradiation under both aerobic and anaerobic conditions, and the yield was approximately 10-fold higher under the latter conditions. The independently generated radical allows for the availability of pure, sufficient and well-characterized intrastrand cross-link lesion-bearing ODN substrates for future biochemical and biophysical characterizations. This was also the first demonstration that the coupling of radical I with its 5' neighboring guanine can occur in the presence of molecular oxygen, suggesting that the formation of this and other types of intrastrand cross-link lesions might have important implications in the cytotoxic effects of ROS.

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Figures

Scheme 1

Scheme 1

Formation of radical I and its coupling with neighboring guanine.

Figure 1

Figure 1

HPLC traces for the separations of single-stranded d(ACGTGYCGTGAT), where Y is the radical precursor, before (a) and after (b) 254 nm irradiation under anaerobic conditions. ‘XL’ (the 22.8 min fraction) represents d(ACGTG∧ mCCGTGAT).

Figure 2

Figure 2

Product-ion spectrum of the electrospray-produced [M−3H]3− ion (m/z 1223.5) of d(ACGTG∧ mCCGTGAT) (the 22.8 min fraction in Figure 1b).

Figure 3

Figure 3

HPLC traces for the separation of nuclease P1 and alkaline phosphatase digestion mixture of d(ACGTG∧ mCCGTGAT) (the 22.8 min fraction in Figure 1b): (a) injection of the digestion mixture; (b) co-injection of digestion mixture and 4.5 nmol d(G∧ mC) standard.

Figure 4

Figure 4

LC-MS/MS results for the injection of d(mC∧ G) and d(G∧ mC) (400 fmol each). Shown are SICs for the m/z 569→275 (a) and m/z 569→471 transitions (b); product-ion spectra of the ions of m/z 569 for the fractions eluting at 20.2 min [(c), corresponding to d(mC∧ G)] and 30.3 min [(d), corresponding to d(G∧ mC)].

Scheme 2

Scheme 2

Radical I can couple with both molecular oxygen and adjacent guanine.

Scheme 3

Scheme 3

Possible enzymic digestion products and the m/z values of their [M + H]+ ions.

Figure 5

Figure 5

LC-MS/MS for the detection of cross-link lesion from the γ irradiation of self-complementary duplex ODN d(mCG)7. The irradiation mixture was digested by nuclease P1, CIP, and phosphodiesterases I and II before LC-MS analysis, and the mass spectrometer was set to monitor the fragmentation of the ion of m/z 569, which is the [M+H]+ ions of the cross-link lesion d(G∧ mC) and d(mC∧ G). Plotted are the SICs for the m/z 569→275 transition with the injections of the digestion mixtures of (a) 1 nmol ODN irradiated with γ-rays in the presence of saturated O2; (b) 1 nmol ODN irradiated with γ-rays in the presence of air; and (c) 0.5 nmol ODN irradiated with γ-rays under anaerobic conditions.

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