Dehydration, deamination and enzymatic repair of cytosine glycols from oxidized poly(dG-dC) and poly(dI-dC)

Abstract

Cytosine glycols (5,6-dihydroxy-5,6-dihydrocytosine) are initial products of cytosine oxidation. Because these products are not stable, virtually all biological studies have focused on the stable oxidation products of cytosine, including 5-hydroxycytosine, uracil glycols and 5-hydroxyuracil. Previously, we reported that the lifetime of cytosine glycols was greatly enhanced in double-stranded DNA, thus implicating these products in DNA repair and mutagenesis. In the present work, cytosine and uracil glycols were generated in double-stranded alternating co-polymers by oxidation with KMnO4. The half-life of cytosine glycols in poly(dG-dC) was 6.5 h giving a ratio of dehydration to deamination of 5:1. At high substrate concentrations, the excision of cytosine glycols from poly(dG-dC) by purified endonuclease III was comparable to that of uracil glycols, whereas the excision of these substrates was 5-fold greater than that of 5-hydroxycytosine. Kinetic studies revealed that the V(max) was several fold higher for the excision of cytosine glycols compared to 5-hydroxycytosine. In contrast to cytosine glycols, uracil glycols did not undergo detectable dehydration to 5-hydroxyuracil. Replacing poly(dG-dC) for poly(dI-dC) gave similar results with respect to the lifetime and excision of cytosine glycols. This work demonstrates the formation of cytosine glycols in DNA and their removal by base excision repair.

Figure 1.

Formation and decomposition of cytosine glycols. Cytosine (1) was oxidized to cytosine glycols (2) by KMnO₄ (Reaction I; Figure 1). Cytosine glycols (2) decomposed by either dehydration to 5-hydroxycytosine (3; Reaction II) or deamination to uracil glycols (4; Reaction III). During acid hydrolysis of DNA, cytosine glycols (2) and uracil glycols (4) are converted to 5-hydroxycytosine (3) and 5-hydroxyuracil (5), respectively (Reactions II and IV; Figure 1). Thus, the amount of 5-hydroxycytosine obtained by acid hydrolysis corresponds to the sum of cytosine glycols (2) and 5-hydroxycytosine (3), whereas the amount of 5-hydroxyuracil (5) corresponds to the sum of uracil glycols (4) and 5-hydroxyuracil (5).

Figure 2.

Analyses of cytosine oxidation products. (a) HPLC/EC analysis of 5-hydroxycytosine (3) and 5-hydroxyuracil (5). Top chromatogram–standard compounds; middle chromatogram–freshly oxidized poly(dG-dC) subjected to acid hydrolysis; bottom chromatogram–heat treated oxidized poly(dG-dC) subjected to acid hydrolysis. Products 3 and 5 were detected by electrochemical detection with the oxidation potential at 75 mV and 350 mV, respectively. (b) GC/MS analysis of 5-hydroxycytosine (3), 5-hydroxyuracil (5) and uracil glycols (4a and 4b). The samples were prepared from polymers by either acid hydrolysis or incubation with Endo III followed by trimethylsilylation of the resulting nucleobases. The most abundant ion in the mass spectrum was chosen for selective ion monitoring (molecular ion −15 amu, unless indicated): 5-hydroxycytosine (3, m/z 328); 5-hydroxyuracil (6, m/z 329); trans uracil glycol (4a, m/z 245, ion fragment) and cis uracil glycol (4b, m/z 245, ion fragment). The peak at 27 min was an impurity. c) Quantitation of 5-hydroxycytosine (3), released from oxidized poly(dG-dC) by Endo III, was achieved by GC/MS analysis with selective ion monitoring. The amount of 5-hydroxycytosine was determined from the ratio of natural product released by Endo III to the corresponding isotopically labeled 5-hydroxycytosine (+3 amu), which was added before the addition of enzyme. The chromatogram depicts the excision of the 5-hydroxycytosine (3) from freshly oxidized poly(dG-dC) (left) and heat-treated polymer (right).

Figure 3.

Thermal decomposition of cytosine glycols in oxidized poly(dG-dC). Decomposition was carried out in phosphate buffer (25 mM, pH 7.0) containing 0.15 NaCl and 1 mM EDTA. Analysis of cytosine glycols (solid circles) and uracil glycols (solid squares) was carried out by acid hydrolysis and HPLC/EC. The dashed line represents the best fit of data to an exponential function [y = y₀ + be (−t/k)], where y₀ and y are the yield of product at time zero and at specific times of incubation (t), respectively, k is the rate of decomposition or growth and b is a constant. From these analyses, the rate of decomposition of cytosine glycols was −0.11 h⁻¹ whereas the growth of uracil glycols was 0.10 h⁻¹ (n = 7; r² ≥ 0.94). Repeated experiments gave similar rates of decomposition and growth. (b) Top panel: decomposition of cytosine glycols as a function of pH (5–8) in phosphate buffer (25 mM) at a fixed concentration of NaCl (0.15 M); bottom panel: decomposition of cytosine glycols as a function of salt concentration (0.15–2 M) in phosphate buffer at pH 7.0 (25 mM, pH 7.0). Rates were estimated from the best fit of data to the above exponential function (n = 7; r² ≥ 0.97).

Figure 4.

Plots of reaction velocity (v) vs substrate concentration. The substrate was either cytosine glycols (solid circles) or 5-hydroxcytosine (open circles) within freshly oxidized poly(dG-dC) or freshly oxidized and then heated polymer, respectively. The red line represents the best fit of data to an exponential function.