Book of Abstracts: Albany 2005
Structure and Stabilities of DNA Duplexes Containing 5-hydroxy Uracil Residues
GC to AT transition mutations are the most abundant base substitution mutation observed in aerobic organisms. It is also the most frequent point mutation resulting from oxidative damage to DNA. Several studies have established that the oxidized cytosine products are the major chemical precursors to this transition mutation. Among the oxidized cytosine products, 5-hydroxy uracil (5-OHU), produced by the oxidative deamination of cytosine, shows the highest mutation frequency. Although the chemical structure of 5-OHU shows it would form normal Watson-Crick type base pairing with adenines, ab initio calculations, NMR, and UV melting experiments reveal that 5-OHU can form stable base pairs with all four standard DNA bases. Results from the ab initio calculations show that 5-OHU forms the most stable pairing with G, and the least stable pairing with C. Using proton and phosphorus NMR spectroscopy, we are studying the structures and dynamics of DNA duplexes containing 5-OHU residues. These experiments were done on the self-complementary DNA duplexes, d(CGCXAATTU*GCG)2, where U* is the 5-OHU and X=A, C, G, or T). Helical parameters and the backbone torsional angles calculated for the refined structures, and the observed phosphorus chemical shifts indicate that the presence of the 5-OHU does not cause any significant perturbations in the DNA backbone. Presence of an additional hydroxyl group in the 5-OHU introduces additional hydrogen bond donor acceptor groups in the major groove of the DNA, and that could serve as the target recognition site for DNA repair enzymes. Among the DNA repair enzymes that repair 5-OHU damages in DNA, the 37-kDa DNA glycosylase NEIL-2 shows the highest activity. Using mutation studies, we have determined the 5-OHU binding region of NEIL-2 to be a 42-residue segment near the C-terminal. The amino acid sequence analysis suggests that a zinc finger motif could be formed in this region. Using site-directed mutagenic studies, we show that this putative zinc finger motif is essential for the enzyme activity, DNA binding, and the structural integrity of NEIL-2. We are probing the solution structure of this zinc finger motif, in isolation and in complex with DNA duplex containing 5-OHU residue.
Department of Human Biological Chemistry & Genetics