NMR structural studies on multi-stranded DNA formed by guanine -rich sequences and duplex DNA containing a carcinogenic benzo[a]pyrene guanine adduct flanked by methylated cytosine.

摘要

We use Nuclear Magnetic Resonance (NMR) spectroscopy to study the structure of DNA in solution, including multi-stranded topologies formed by guanine-rich sequences and Watson-Crick DNA duplexes containing carcinogenic adducts. In chapter 2, we show that the three-repeat human telomeric DNA sequence folds into a unique asymmetric dimeric quadruplex, in which the G-tetrad core involves all three G-tracts of one strand and the 3'-end G-tract of a second strand. In addition, the three-repeat human telomeric sequence can also associate with the short single-repeat human telomeric sequence to form a hetero-dimeric quadruplex with the same core topology. Chapter 3 describes the structure of a dimeric quadruplex formed by the d(GAGCAGGT) sequence, which is generated through stacking of G·G·G·G, direct G·C·G·C and slipped A·T·A·T tetrads. The two mixed tetrads involve recognition of Watson-Crick G·C and A·T pairs along their major groove edges, with pairing occurring between rather than within hairpin subunits of this quadruplex. In chapter 4, we report that the d(GGGAGGTTTGGGAT) sequence dimerizes into a quadruplex containing a V-shaped scaffold and a A·(G·G·G·G) pentad. Taken together, the results in chapters 2-4 significantly broaden our knowledge of structural features of G-quadruplexes, including base pairing alignments, loop topologies and strand orientations. In chapter 5, we study the structure of the d(GGAAGGTTTGGGAT) sequence, which differs from the sequence studied in chapter 4 by only a single adenine-for-guanine substitution in the third position. This sequence dimerizes to form a novel structure, in which each arrowhead-like segment contains two short parallel-stranded helices. The central core is composed of a pair of symmetry-related stacked G·G·G base triple platforms linked by two rather than three phosphodiester bonds.;DNA duplexes containing stereoisomeric [BP]dG adducts, the products of guanine reacting with active epoxide metabolites of carcinogenic benzo[ a]pyrene, are studied in chapter 6. Correlated to the fact that CpG dinucleotides are highly methylated at the 5-position of cytosines in human tissues, my structural study demonstrates that the methylation of a flanking cytosine has a profound effect on the conformational alignments of covalently-bound adducts at the lesion site. In the case of the S-(-) -trans-anti-[BP]G stereoisomer positioned within the meC-[BP]G-C sequence context, such methylation switches the adduct alignment from a minor-groove to an intercalative alignment. These conformations are sufficiently distinct to be differentially targeted by the cellular repair machinery.