Jaruga Pawel, Theruvathu Jacob, Dizdaroglu Miral, Brooks Philip J
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, MD 22777, USA.
Nucleic Acids Res. 2004 Jun 23;32(11):e87. doi: 10.1093/nar/gnh087. Print 2004.
8,5'-cyclopurine-2'-deoxynucleosides in DNA are repaired by nucleotide-excision repair, and act as strong blocks to DNA polymerases, RNA polymerase II and transcription factor binding. Thus, it is important to accurately determine the level of these lesions in DNA. There is controversy in the literature regarding the ability of different enzymes to release these compounds from oligodeoxynucleotides or DNA. We used liquid chromatography/mass spectrometry (LC/MS) to investigate the ability of several enzymes to release (5'S)-8,5'-cyclo-2'-deoxyadenosine [(5'S)-cdA] from dinucleotides and oligodeoxynucleotides and from DNA. The data show that (5'S)-cdA is completely released from DNA by hydrolysis with nuclease P1, snake venom phosphodiesterase and alkaline phosphatase. The identity of the normal nucleoside 5' to the (5'S)-cdA had a significant effect on its release. Using LC/MS, we also showed that the levels of (5'S)-cdA were within an order of magnitude of those of 8-hydroxy-2'-deoxyguanosine, and three times higher than those of 8-hydroxy-2'-deoxyadenosine in pig liver DNA. Different DNA isolation methods affected the levels of the latter two lesions, but did not influence those of (5'S)-cdA. We conclude that (5'S)-cdA can be completely released from DNA by enzymic hydrolysis, and the level of (5'S)-cdA in tissue DNA is comparable to those of other oxidatively induced DNA lesions.
DNA中的8,5'-环嘌呤-2'-脱氧核苷通过核苷酸切除修复进行修复，并对DNA聚合酶、RNA聚合酶II和转录因子结合起到强大的阻碍作用。因此，准确测定DNA中这些损伤的水平非常重要。关于不同酶从寡脱氧核苷酸或DNA中释放这些化合物的能力，文献中存在争议。我们使用液相色谱/质谱联用（LC/MS）来研究几种酶从二核苷酸、寡脱氧核苷酸以及DNA中释放(5'S)-8,5'-环-2'-脱氧腺苷[(5'S)-cdA]的能力。数据表明，通过核酸酶P1、蛇毒磷酸二酯酶和碱性磷酸酶水解，(5'S)-cdA可从DNA中完全释放出来。(5'S)-cdA 5'端正常核苷的身份对其释放有显著影响。使用LC/MS，我们还表明，在猪肝DNA中，(5'S)-cdA的水平与8-羟基-2'-脱氧鸟苷的水平在一个数量级内，且是8-羟基-2'-脱氧腺苷水平的三倍。不同的DNA分离方法会影响后两种损伤的水平，但不影响(5'S)-cdA的水平。我们得出结论，(5'S)-cdA可通过酶促水解从DNA中完全释放出来，并且组织DNA中(5'S)-cdA的水平与其他氧化诱导的DNA损伤水平相当。
