Bisulfite-induced Deamination Of Cytosine And Its Hydrolyzed Derivatives Mechanism Theory

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.