Chen Xin, Fleming Aaron M, Muller James G, Burrows Cynthia J
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, U.S.A.
New J Chem. 2013 Nov 1;37(11):3440-3449. doi: 10.1039/C3NJ00418J.
8-Oxo-7,8-dihydro-2'-deoxyguanosine (dOG), a well-studied oxidation product of 2'-deoxyguanosine (dG), is prone to facile further oxidation forming spiroiminodihydantoin 2'-deoxyribonucleoside (dSp) in the nucleotide pool and in single-stranded oligodeoxynucleotides (ODNs). Many methods for quantification of damaged lesions in the genome rely on digestion of DNA with exonucleases or endonucleases and dephosphorylation followed by LC-MS analysis of the resulting nucleosides. In this study, enzymatic hydrolysis of dSp-containing ODNs was investigated with snake venom phosphodiesterase (SVPD), spleen phosphodiesterase (SPD) and nuclease P1. SVPD led to formation of a dinucleotide, 5'-d(Np[Sp])-3' (N = any nucleotide) that included the undamaged nucleotide on the 5' side of dSp as the final product. This dinucleotide was a substrate for both SPD and nuclease P1. A kinetic study of the activity of SPD and nuclease P1 showed a sequence dependence on the nucleotide 5' to the lesion with rates in the order dG>dA>dT>dC. In addition, the two diastereomers of dSp underwent digestion at significantly different rates with dSp1>dSp2; nuclease P1 hydrolyzed the 5'-d(Np[Sp1])-3' dinucleotide two- to six-fold faster than the corresponding 5'-d(Np[Sp2])-3', while for SPD the difference was two-fold. These rates are chemically reasoned based on dSp diastereomer differences in the vs. glycosidic bond orientation. A method for the complete digestion of dSp-containing ODNs is also outlined based on treatment with nuclease P1 and SVPD. These findings have significant impact on the development of methods to detect dSp levels in cellular DNA.
8-氧代-7,8-二氢-2'-脱氧鸟苷(dOG)是一种经过充分研究的2'-脱氧鸟苷(dG)氧化产物,在核苷酸池和单链寡脱氧核苷酸(ODN)中容易进一步氧化形成螺亚胺二氢尿嘧啶2'-脱氧核糖核苷(dSp)。许多用于定量基因组中损伤位点的方法依赖于用核酸外切酶或核酸内切酶消化DNA并进行去磷酸化,然后对所得核苷进行液相色谱-质谱分析。在本研究中,用蛇毒磷酸二酯酶(SVPD)、脾磷酸二酯酶(SPD)和核酸酶P1研究了含dSp的ODN的酶促水解。SVPD导致形成二核苷酸5'-d(Np[Sp])-3'(N = 任何核苷酸),其作为最终产物在dSp的5'侧包含未受损的核苷酸。该二核苷酸是SPD和核酸酶P1的底物。对SPD和核酸酶P1活性的动力学研究表明,其活性对损伤位点5'侧的核苷酸具有序列依赖性,速率顺序为dG>dA>dT>dC。此外,dSp的两种非对映异构体以明显不同的速率进行消化,dSp1>dSp2;核酸酶P1水解5'-d(Np[Sp1])-3'二核苷酸的速度比相应的5'-d(Np[Sp2])-3'快两到六倍,而对于SPD,差异为两倍。这些速率是基于dSp非对映异构体在糖苷键取向方面的差异从化学角度推断出来的。还概述了一种基于用核酸酶P1和SVPD处理来完全消化含dSp的ODN的方法。这些发现对检测细胞DNA中dSp水平的方法的开发具有重大影响。