Synthesis of fluorinated analogs of oxidative DNA lesions and their use to probe features of recognition and repair by base excision repair glycosylases

Damage to DNA occurs through various sources, both exogenous and endogenous. Base lesions resulting from the oxidation of guanine include 8-oxo-7,8-dihydroguanine (OG), guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp). These modified bases can form mismatches and lead to a G → C and G → T transversion mutations in DNA. Fortunately, these lesions are recognized and removed by DNA repair glycosylases found in all organisms. This work explores the features of recognition and repair of these oxidized guanine lesions by a diverse set of base excision glycosylases (MutY, Fpg, hOGG1, Nei, hNEIL1) using synthesized 2' fluorinated nucleosides such as 2'-fluoro-adenosine (FA), 2'-fluoro-8-oxo-guanosine (FOG), 2'-fluoro-guanidinohydan-toin (FGh), and 2'-fluoro-spiroiminodihydantoin (FSp).;FA and FOG nucleosides in both ribo and arabino sugar conformations have been synthesized and incorporated into oligonucleotide duplexes. Furthermore, oxidation of FOG within an oligonucleotide strand yielded FGh and FSp, both with the ribo and arabino sugar conformation. FA is not a substrate for the adenine glycosylase MutY. However, FA with the fluorine in arabino conformation binds to MutY about 4 fold tighter than the fluorine in the ribo conformation. Similar studies with FOG, FGh, and FSp analogues showed Fpg and its functional homologous human endonuclease VIII-like DNA glycosylase (hNEIL1) can recognize all three lesion containing oligos efficiently. FOG is not a substrate for Fpg, Nei and hNEIL1, and FGh and FSp have increased resistance to cleavage compared to Gh and Sp with all these glycosylases. The sugar conformation of F-nucleotide influences the extent of cleavage as well as binding affinity of the glycosyalse is an important factor to affect the recognition and repair of these lesions.;Duplex stability with unmodified and modified nucleosides have been evaluated by melting temperature measurements, and results revealed duplex destabilization caused by introducing these lesions may be a factor leading to improved recognition of these analogues. X-ray crystallography studies about Fpg and NEIL1 bound to DNA duplexes containing these analogues are ongoing.;In addition, transition state analogue 1-aza-deoxyribose (1N) was synthesized and the crystal structures of Bacillus stearothermophilus (Bs) MutY bound to DNA duplexes containing 1N were determined. These structures provide the first view of an intermediate step of the MutY reaction and necessitate modification of previously proposed models for the mechanism of MutY.