Formation and characterization of oxidative intrastrand crosslink lesions of DNA.

摘要

Reactive oxygen species (ROS) can be produced by both endogenous and exogenous processes and they can damage biological molecules including nucleic acids. It was shown that X- or gamma-ray irradiation of DNA, which produces hydroxyl radical (•OH), can lead to the formation of intrastrand crosslink lesions where the neighboring nucleobases in the same DNA strand are covalently bonded.; In Chapter 2, I showed that Pyrex-filtered UV light irradiation of d( BrCA) ("BrC" represents 5-bromocytosine) gave rise to three types of intrastrand crosslink products, that is, d(C[5-N 6]A), d(C[5-2]A), and d(C[5-8]A), where the C5 carbon atom of cytosine is covalently bonded to the N6 nitrogen atom, C2, and C8 carbon atoms of adenine, respectively. Furthermore, I demonstrated by LC-MS/MS that the UV irradiation of a BrC-containing duplex oligodeoxyribonucleotide (ODN) led to the formation of five cross-link products, that are, C[5-N 6]A, C[5-2]A, C[5-8]A, A[2-5]C, and A[8-5]C, under both aerobic and anaerobic conditions.; In Chapter 3, I showed for the first time that the treatment of calf thymus DNA with Cu(II)/H2O2/ascorbate could lead to the formation of a guanine-thymine intrastrand cross-link lesion, i.e., G[8-5m]T. LC-MS/MS quantification results showed that the yield of G[8-5m]T was approximately 3 orders of magnitude lower than those of commonly observed single-base lesions, that is, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 5-(hydroxymethyl)-2'-deoxyuridine (HmdU), and 5-formyl-2'-deoxyuridine (FodU).; In Chapter 4, I described a new approach for the highly sensitive detection of FodU, one of the most important single-nucleobase lesions induced by ROS. I derivatized FodU with Girard reagent T to form a hydrazone conjugate, which enabled the facile ionization of the resulting conjugate by ESI-MS. I also showed that the combination of the derivatization with LC-MS/MS on a linear ion-trap mass spectrometer could allow for the quantification of FodU at a detection limit of 3-4 fmol, which is ∼20 fold better than for the direct analysis of the underivatized compound.; In Chapter 5, I assessed quantitatively the formation of a guanine-cytosine (G[8-5]C) intrastrand crosslink lesion in HeLa-S3 cells upon exposure to gamma-rays. The yield of the G[8-5]C crosslink was 0.037 lesions per 109 nucleosides per Gy, which was approximately 300 times lower than that of FodU (0.011 lesions per 106 nucleosides per Gy) under identical exposure conditions. I further constructed a single-stranded M13 genome harboring a site-specifically incorporated G[8-5]C lesion and developed a novel mass spectrometry-based method for interrogating the products emanating from the replication of the genome in Escherichia coli (E. coli) cells. The results demonstrated that G[8-5]C considerably blocked DNA replication as represented by a 20% bypass efficiency, and the lesion was significantly mutagenic in vivo, resulting in 8.7% G → T and 1.2% G → C transversion mutations.