Non-enzymatic depurination of nucleic acids: factors and mechanisms

Abstract

Depurination has attracted considerable attention since a long time for it is closely related to the damage and repair of nucleic acids. In the present study, depurination using a pool of 30-nt short DNA pieces with various sequences at diverse pH values was analyzed by High Performance Liquid Chromatography (HPLC). Kinetic analysis results showed that non-enzymatic depurination of oligodeoxynucleotides exhibited typical first-order kinetics, and its temperature dependence obeyed Arrhenius' law very well. Our results also clearly showed that the linear relationship between the logarithms of rate constants and pH values had a salient point around pH 2.5. Interestingly and unexpectedly, depurination depended greatly on the DNA sequences. The depurination of poly (dA) was found to be extremely slow, and thymine rich sequences depurinated faster than other sequences. These results could be explained to some extent by the protonation of nucleotide bases. Moreover, two equations were obtained based on our data for predicting the rate of depurination under various conditions. These results provide basic data for gene mutagenesis and nucleic acids metabolism in acidic gastric juice and some acidic organelles, and may also help to rectify some misconceptions about depurination.

Figure 1. Time courses of non-enzymatic depurination.

(a) Quantitative analysis of purines released from N30 at 37°C. (•) pH 1.6; (▴) pH 2.0; (○) pH 2.5; () pH 3.0. Reaction buffers for depurination contained 50 mM sodium phosphate. Samples were prepared by collecting aliquots of the solution in each time period. The percentages of depurination were obtained by averaging the percentages of the release of adenine and guanine. The plot shows the average of three individual experiments. (b) Depurination of calf thymus DNA at 37°C and pH 1.6 reported by Tamm et al. . (○) Adenine; (•) Guanine.

Figure 2. Plots of rate constants (k, s⁻¹) for the depurination of N30 as a function of pH.

(•) 37°C, the slopes are −0.721 at pH 1.0−2.5 and −0.975 at pH 2.5−7.1; (○) 65°C, the slopes are −0.694 at pH 0.5−2.5 and −0.983 at pH 2.5−7.1.

Figure 3. Arrhenius plot for depurination from N30 at different pH values.

Rate constants (k, s⁻¹) of depurination were a function of absolute temperature (T). (○) pH 1.0; (•) pH 2.0; (•) pH 4.1.

Figure 4. The effect of salts on depurination from N30.

(a) Depurination in the NaCl buffer (pH 1.4); (b) Depurination in the MgCl₂ buffer (pH 1.4); (c) Depurination in the NaCl buffer (pH 5.9); (d) Depurination in the MgCl₂ buffer (pH 5.9). The pH values of the buffers were adjusted to 1.4 (±0.1) or 5.9 (±0.2) with an aqueous solution of HCl. The reaction was performed for 60 min at 37°C (pH 1.4) or 24 h at 80°C (pH 5.9) to limit the percentages of depurination less than 20%. In each chart, the depurination rate constant of N30 in the pure HCl buffer was served as the reference.

Figure 5. Difference of various sequences for depurination.

() Adenine; (□) Guanine. In each reaction condition, the half-lives of N30 (depurination of adenine) were served as the references. The sequences used for depurination are listed in Table 2. The insert is the enlarged view at pH 5.1, 80°C.

Figure 6. Difference between single-stranded and double-stranded DNA for depurination at pH 5.1, 60°C.

(a) Time courses of depurination of M13 ssDNA and dsDNA. (○) M13 dsDNA; (•) M13 ssDNA. (b) The rate constants (k) of several kinds of DNA including ssDNA (M13 ssDNA and N30) and dsDNA (M13 dsDNA, Salmon sperm DNA and Lambda DNA). T _m analysis showed that M13 dsDNA kept duplex form below 70°C at pH 5.1.

Figure 7. Protonation mechanism of depurination.

Adenine is monoprotonated on N7 under acidic conditions, finally forming a neutral adenine and a deoxyribose as the end products. As the pH lowers, the double protonation process occurs by the attack of excessive H⁺ on N3.