摘要
胞嘧啶(Cyt)及其C5-甲基化衍生物(5-MeCyt)广泛存在于生物体系中,这些衍生物在DNA链上对抑制基因表达、维持染色体结构、X染色体失活、基因印记及肿瘤的发生等有着重要影响。而且,这些衍生物在DNA链上的去甲基化(或去氨基)生成胞嘧啶(或胸腺嘧啶)将引发基因突变,导致诸多疾病的发生。因此,在临床上对DNA中胞嘧啶甲基化位置的准确识别尤为重要。目前,亚硫酸氢盐是一种广泛使用的DNA甲基化检测试剂,但由于该检测技术易受外界环境影响,使得DNA片段经亚硫酸氢盐处理后,部分Cyt未转化为尿嘧啶,而部分5-MeCyt却转化为胸腺嘧啶,从而导致DNA甲基化密度的错误估计。本论文采用量子化学双水平计算方法和组合方法,以揭示该检测技术错误估计DNA甲基化密度的深层次原因。为此,主要开展以下三个方面的研究工作,并得到了与实验现象基本吻合而又十分有意义的结论,为实验工作者探索基于亚硫酸氢盐检测DNA甲基化最佳条件提供了参考。
5-羟甲基胞嘧啶(5-hmCyt),可能是一个重要的表观遗传学标记,也可能是5-MeCyt去甲基向Cyt转化的中间物。目前,氧化的亚硫酸氢盐测序是首次以单碱基分辨率对5-hmCyt进行定量测序的方法,该方法的主要步骤为：首先将5-hmCyt氧化为5-羧基胞嘧啶(5-caCyt)和5-甲酰基胞嘧啶(5-fCyt),然后经亚硫酸氢盐处理,脱氨基形成尿嘧啶。目前认为5-caCyt和5-fCyt在经亚硫酸氢盐处理前,将经历去羧基和去甲酰基直接转化为Cyt,而最近实验却表明5-caCyt和5-fCyt转化为Cyt的产率极低,由此设想,未转化的5-caCyt和5-flCyt经亚硫酸氢盐处理后是否可以发生去氨基反应,如果是,这两类反应是否存在竞争。为此,主要开展以下第四个方面的研究工作,并得到了一些新的结论,为实验研究亚硫酸氢盐DNA甲基化检测技术提供了新视角。
一、利用CBS-QB3组合方法,研究了Cyt(5-MeCyt)在酸性条件下形成质子化异构体的稳定性以及质子化异构体间的相互转化机理,并比较了它们在酸性和中性条件下与HSO3-加成反应的差异,讨论了反应势能面及反应力等信息,同时采用从静电势导出原子净电荷的ChelpG法预测了亲核/亲电反应位点。研究结果表明Cyt(5-MeCyt)在N3和O2位置形成的质子化异构体CytN3+(5-MeCytN3+)和Cyt2t+(5-MeCyt2t+)较其它位置稳定；不管在气相还是在液相,质子化作用显著降低了Cyt(5-MeCyt)各加成反应通道的活化自由能；在气相中Cyt2t+(5-MeCyt2t+)极易与HSO3-发生加成反应,而在液相中CytN3+(5-MeCytN3+)与HSO3-是最有可能发生的反应通道；C5-甲基化使得Cyt的各加成反应趋势减弱；C5-甲基化通道,其加成产物存在三个异构体,除了形成顺式异构体(cis-isomer)和反式异构体(trans-isomer)外,还形成了第三种异构体(third-isomer),且顺式异构体极易向第三种异构体转化。
二、采用MP2/6-311++G(3df,3pd)//B3LYP/6-311++G(d,p)双水平计算方法及CBS-QB3组合方法,研究了CytN3+与HSO3-加成产物(CytN3+-SO3-)的直接水解和HSO3-诱导水解去氨基反应机理,并对比了这两类机理的差异,同时考察了温度与各通道假一级速率常数的关系以及讨论了亚硫酸氢根浓度对反应速率的影响。研究结果表明,HSO3-的参与明显降低了水解反应的自由能垒,直接水解反应的假一级速率常数(k')明显小于HSO3-诱导水解去氨基的k',且HSO3-诱导水解反应是最有可能存在的机理,其计算的假一级速率常数k'(1.99～3.81×10-5s-1)与实验测定的假一级速率常数(26.20×10-5s-1)接近。此外,研究结果还表明,这两类反应速控步骤的假一级常数与温度呈线性关系,且在给定温度下,这两类反应速率的比值与HSO3-浓度呈正相关。
三、采用MP2/6-311++G(3df,3pd)//B3LYP/6-311G(d,p)双水平计算方法考察了HSO3-与5-MeCytN3+加成产物(5-MeCytN3+-SO3-)的顺反异构体与HSO3-水解去氨基机理,并比较了顺反异构体与HSO3-水解反应机理的差异,进一步讨论了异构体浓度与反应速率的关系。研究结果表明,DNA片段经亚硫酸氢盐处理后,5-MeCytN3+-SO3-异构体均有可能转化为胸腺嘧啶,且5-MeCytN3+-SO3-异构体水解反应速率之比与其相应浓度的比值相等。
四、采用MP2/6-311++G(3df,3pd)//B3LYP/6-311++G(d,p)双水平计算方法对比研究了HSO3-诱导5-caCytN3+-SO3-和5-O+fCytN3+-SO3-水解去氨基反应机理的差异,并与HSO3-诱导CytN3+-SO3-水解去氨基的反应机理进行了比较,同时对各通道速控步骤自由能垒进行了讨论。其研究结果表明,5-caCytN3+-SO3-在HSO3-存在下,水解去氨基反应的自由能垒明显低于5-O+fCytN3+-SO3-和CytN3+-SO3-通道,因此在亚硫酸氢盐条件下,5-caCyt去氨基反应极有可能发生。同时,HSO3-诱导5-O+fCytN3+-SO3-水解去氨基反应速控步骤的自由能垒与同水平下获得的CytN3+-SO3-的自由能垒极为接近,表明5-fCyt的去氨基反应在HSO3-存在下也有可能发生。
Cytosine (Cyt) and its C5-methyl derivative (5-MeCyt) are found in biological systems, and these derivatives have significant effects on the genomic expression, the structure of chromosome, activation of the X chromosome, genomic imprinting, and producing of tumors. Meanwhile, DNA methylation is believed to cause about one-third of all transition mutations, and it is responsible for human genetic diseases. Thus, determination of the position of DNA methylation has become clinically important prerequisite in understanding the diseases and embryonic developments. Now, the bisulfite genomic sequencing is believed to be the most common and classical approach for determining of DNA methylation. However, the bisulfite genomic sequencing is very easy to be influenced by external environment. Unmethylated Cyt treated with bisulfite is either converted to uracil or failes to be converted and remains as Cyt, and the5-MeCyt either does not undergo conversion or is inappropriately converted to thymine. Both types of conversion would lead to the erroneous estimates of methylation densities. To understand the profound reason of the inappropriate conversion mechanism, three aspects in this dissertation are as follows:
5-hydroxymethylcytosine (5-hmCyt) is an epigenetic DNA mark, and may be an intermediate in active DNA demethylation. Present oxidative bisulfite sequencing (oxBS-Seq) is the first method for quantitative mapping of5-hmCyt in genomic DNA at single-nucleotide resolution. Selective oxide of5-hmCyt to5-carboxycytosine (5-caCyt) and5-formylcytosine (5fCyt) enables bisulfite conversion of them to uracil. However, recent experiments reported that the yield of Cyt from a decarboxylation of5-caCyt and a deformylation of5-fCyt was very low. These intriguing phenomena inspire us to question whether the rest of5-caCyt and5-fCyt by bisulfite treatment may result in deamination to give uracil. If so, whether there is competition between them treated with bisulfite? Therefore, the fourth aspect has been carried out, and some new useful conclusions are obtained. These results give a possible new insight on the5-caCyt and5-fCyt under typical bisulfite conditions.
1. The stability and isomerization mechanisms of protonated Cyt and5-MeCyt isomers have been investigated by CBS-QB3composite approach; the difference of reaction trend in Cyt+HSO3-reactions is taken into account under neutral and acidic conditions; the potential energy surfaces and the evolution of the reaction force are discussed. Meanwhile, the site of electrophilicity or nucleophilicity index has been predicted by ChelpG procedure. Our calculations indicate the other protonated forms are obviously less stable than those of N3, O2-protonated forms (CytN3+and Cyt2t+); the proton-catalyzed process in reactions of Cyt+HSO3-and5-MeCyt+HSO3-is more favorable than their non-catalytic process both in the gas and aqueous phases; in the gas phase Cyt2t+path is the most likely to occur, whereas in the aqueous phase CytN3+path is the most feasible mechanism; compared with Cyt, the trend of the C5-methylation reaction with HSO3-group, in neutral and acidic conditions, tend to be decreased; apart from cis-isomer and trans-isomer, the third-isomer has been found in the reactions of neutral and protonated5-MeCyt with HSO3-group. The transformation of the third-isomer from cis-isomer can occur easily.
2. Two distinct groups of mechanisms for the direct and HSO3--induced hydrolytic deamination reactions have been explored by CBS-QB3composite approach and MP2/6-311++G(3df,3pd)//B3LYP/6-311++G(d,p) level, respectively. Meanwhile, the difference between the direct hydrolytic and HSO3--induced hydrolytic deamination reactions, the relationship of the pseudo-first-order rate constant and temperature, as well as the effect of bisulfite concentration on the deamination rate have been explored. The calculated results show that the activation free energies of the HSO3--induced reaction are significantly decreased; the pseudo-first-order rate constant (k') for direct hydrolysis is obviously smaller than that of HSO3--induced hydrolytic deamination, which is the most plausible mechanism, where the calculated the k'(1.99～3.81×10-5s-1) is in close proximity to the experimentally determined the pseudo-first-order rate constant (26.2×10-5s-1). Furthermore, the results also manifest that there is positive correlation between the k' and temperature, and the ratio of reaction rates between direct hydrolysis reaction and HSO3--induced hydrolytic deamination increases with the increase of the bisulfite concentration at a given temperature.
3. HSO3--induced hydrolytic deamination of5-MeCytN3+-SO3-isomers has been explored by MP2/6-311++G(3df,3pd)//B3LYP/6-311G(d,p) level. Meanwhile, the difference between cis-isomer and trans-isomer reaction mechanism has been compared. Furthermore, the relationship of the5-MeCytN3+-SO3-isomers concentration and the deamination rate has been investigated as well. The calculated results show that the difference in free barrier is small in these isomer paths, manifesting that these isomers may be contributed to the conversion of5-MeCyt to thymine through bisulfite catalysis. In addition, the results also illustrate that the reaction rate of each isomer is dependent on the concentration of the isomer.
4. The hydrolytic deamination mechanisms of5-caCyt and5-fCyt in bisulfite conditions have been explored at the MP2/6-311++G(3df,3pd)//B3LYP/6-311++G(d,p) level. The activation free energy (AGs≠=54.16kJ-mol-1) for the hydrolysis deamination of5-caCytN3+-SO3-is much lower than that of the ACs≠of CytN3+-SO3-(100.91kJ-mol-1) under bisulfite conditions, implying that5-caCyt may firstly involve a process of deamination. Meanwhile, the ΔGs≠(103.84kJ·mol-1) for the HSO3--induced hydrolytic deamination of5-O+fCytN3+-SO3-is in close proximity to our previous theoretical data of CytN3+-SO3-, indicating that the deamination of5-fCyt are also likely to occur in the presence of bisulfite.
引文
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