Sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex DNA containing a 5-bromo-2'-deoxyuridine or 5-bromo-2'-deoxycytidine - PubMed (original) (raw)

Yu Zeng et al. Nucleic Acids Res. 2006.

Abstract

The replacement of thymidine with 5-bromo-2'-deoxyuridine (BrdU) is well-known to sensitize cells to ionizing radiation and photoirradiation. We reported here the sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex oligodeoxynucleotides harboring a BrdU or its closely related 5-bromo-2'-deoxycytidine (BrdC). Our results showed that two types of crosslink products could be induced from d(BrCG), d(BrUG), d(GBrU), or d(ABrU); the C(5) of cytosine or uracil could be covalently bonded to the N(2) or C(8) of its neighboring guanine, and the C(5) of uracil could couple with the C(2) or C(8) of its neighboring adenine. By using those crosslink product-bearing dinucleoside monophosphates as standards, we demonstrated, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), that all the crosslink products described above except d(G[N(2)-5]U) and d(G[N(2)-5]C) could form in duplex DNA. In addition, LC-MS/MS quantification results revealed that both the nature of the halogenated pyrimidine base and its 5' flanking nucleoside affected markedly the generation of intrastrand crosslink products. The yields of crosslink products were much higher while the 5' neighboring nucleoside was a dG than while it was a dA, and BrdC induced the formation of crosslink products much more efficiently than BrdU. The formation of intrastrand crosslink products from these halopyrimidines in duplex DNA may account for the photosensitizing effects of these nucleosides.

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Figures

Scheme 1

Scheme 1

Intrastrand crosslink products formed from d(BrUG) and d(ABrU).

Scheme 2

Scheme 2

Proposed mechanisms for the formation of intrastrand crosslink products between uracil and guanine.

Figure 1

Figure 1

HPLC traces for the separation of the aerobic irradiation mixtures of d(BrUG) (a) and d(BrCG) (b).

Figure 2

Figure 2

Positive-ion product-ion spectra of d(U[5-8]G): MS/MS (a), MS3 (b), MS4 (c) and d(G[8-5]U): MS/MS (d), MS3 (e), MS4 (f). The relative collisional energies were 30%.

Figure 3

Figure 3

LC-MS/MS analysis of the standard d(U ^ G) (a) and d(G ^ U) (c) crosslink products and the enzymatic digestion products of the aerobic UVB-irradiated duplex ODN d(ATGGCGBrUGCTAT)/d(ATAGCACGCCAT) (b and d). Shown are the SICs for the monitoring of the m/z 556→538 (a and b) and m/z 556→458 (c and d) transitions.

Figure 4

Figure 4

Positive-ion product-ion spectra of d(C[5-N(2)]G): MS/MS (a), MS3 (d); d(C[5-8]G): MS/MS (b), MS3 (e); and d(G[8-5]C): MS/MS (c), MS3(f). The relative collisional energies were 30%.

Figure 5

Figure 5

LC-MS/MS analysis of the standard d(C ^ G) and d(G ^ C) crosslink products (a) and the enzymatic digestion products of the UVB-irradiated duplex ODN d(ATGGCGBrCGCTAT)/d(ATAGCGCGCCAT) (b). Shown are the SICs for monitoring the m/z 555→261 transition. A portion of SIC for the analysis of the digestion products was plotted in a different scale to better view the formation of the two C ^ G crosslinks [inset in (b)].

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