NMR investigations of strand slippage in CTG repeat expansion and primer-template misalignment in low fidelity DNA replication.

摘要

DNA has been found to adopt unusual structures leading to different types of mutations, which can ultimately cause genetic diseases and cancers. In this thesis, investigations on (i) structural role of CTG repeats in trinucleotide repeat expansion, (ii) primer-template structures in strand slippage during low fidelity replication and (iii) sequence effect of nucleotide downstream of thymine templates on primer-template structures have been carried out using NMR spectroscopy.;CTG repeat is one of the most common triplet repeat sequences that have been found to form slipped-strand structures leading to self-expansion during DNA replication. The lengthening of these repeats causes the onset of neurodegenerative diseases such as myotonic dystrophy. Through designing a series of CTG repeat sequences with high hairpin populations, systematic analysis of imino and methyl proton spectra study has been carried out to investigate the length and structural roles of CTG repeats in affecting the propensity of hairpin formation. Direct NMR evidence has been obtained to support three types of hairpin structures in sequences containing one to ten CTG repeats. The differences in loop structures and extent of interactions observed in the hairpins account for the differences in hairpin formation propensity and explain how slippage occurs that lead to triplet repeat expansion.;In addition, NMR structural investigations have also been carried out to determine solution structures of primer-template models. NMR evidence confirms misalignment can occur in primer-templates upon misincorporation of dNTP opposite a template sequence, leading to bulge formation in the primer-template. Depending on the template sequence, further incorporation of dNTP can bring about either realignment or further stabilization of the primer-template structure. Consequently, either mismatch or deletion errors will occur, leading to base substitution or frameshift mutation. These results imply that DNA sequences do not only play a passive role to store genetic information in the replication process, they also play an active structural role in governing the types of mutation during low-fidelity DNA replication.;Some of the results in this thesis have been reported in the following peer-reviewed journals: (1) Chi, L. M. and Lam, S. L. (2005) Structural roles of CTG repeats in slippage expansion during DNA replication. Nucleic Acids Res, 33, 1604-1617. (2) Chi, L. M. and Lam, S. L. (2006) NMR investigation of DNA primer-template models: structural insights into dislocation mutagenesis in DNA replication. FEBS Lett. , 580, 6496-6500. (3) Chi, L. M. and Lam, S. L. (2007) NMR investigation of primer-template models: structural effect of sequence downstream of a thymine template on mutagenesis in DNA replication. Biochemistry, 46, 9292-9300.