Interconversion of the cis-5 R,6 S- and trans-5 R,6 R-Thymine Glycol Lesions in Duplex DNA

Abstract

Thymine glycol (Tg), 5,6-dihydroxy-5,6-dihydrothymine, is formed in DNA by the reaction of thymine with reactive oxygen species. The 5 R Tg lesion was incorporated site-specifically into 5′-d(G ¹T ²G ³C ⁴G ⁵ Tg ⁶ G ⁷T ⁸T ⁹T ¹⁰G ¹¹T ¹²)-3′; Tg = 5 R Tg. The Tg-modified oligodeoxynucleotide was annealed with either 5′-d(A ¹³C ¹⁴A ¹⁵A ¹⁶A ¹⁷C ¹⁸ A ¹⁹ C ²⁰G ²¹C ²²A ²³C ²⁴)-3′, forming the Tg ⁶• A ¹⁹ base pair, corresponding to the oxidative damage of thymine in DNA, or 5′-d(A ¹³C ¹⁴A ¹⁵A ¹⁶A ¹⁷C ¹⁸ G ¹⁹ C ²⁰G ²¹C ²²A ²³C ²⁴)-3′, forming the mismatched Tg ⁶• G ¹⁹ base pair, corresponding to the formation of Tg following oxidative damage and deamination of 5-methylcytosine in DNA. At 30 °C, the equilibrium ratio of cis-5 R,6 S: trans-5 R,6 R epimers was 7:3 for the duplex containing the Tg ⁶• A ¹⁹ base pair. In contrast, for the duplex containing the Tg ⁶• G ¹⁹ base pair, the cis-5 R,6 S: trans-5 R,6 R equilibrium favored the cis-5 R,6 S epimer; the level of the trans-5 R,6 R epimer remained below the level of detection by NMR. The data suggested that Tg disrupted hydrogen bonding interactions, either when placed opposite to A ¹⁹ or G ¹⁹. Thermodynamic measurements indicated a 13 °C reduction of T _m regardless of whether Tg was placed opposite dG or dA in the complementary strand. Although both pairings increased the free energy of melting by 3 kcal/mol, the melting of the Tg•G pair was more enthalpically favored than was the melting of the Tg•A pair. The observation that the position of the equilibrium between the cis-5 R,6 S and trans-5 R,6 R thymine glycol epimers in duplex DNA was affected by the identity of the complementary base extends upon observations that this equilibrium modulates the base excision repair of Tg [Ocampo-HafallaM. T.; AltamiranoA.; BasuA. K.; ChanM. K.; OcampoJ. E.; CummingsA.Jr.; BoorsteinR. J.; CunninghamR. P.; TeeborG. W. DNA Repair (Amst) 2006, 5, 444−454].