The expansion of trinucleotide repeat tracts (e.g. (CAG)n tracts) has been shown to contribute to genomic instability and has been implicated in the pathogenesis of several neurodegenerative diseases, including Huntington's Disease and Fragile X syndrome.  While the molecular mechanism of this expansion is unknown, the ability of trinucleotide repeat sequences to form non-canonical secondary structures, such as stem-loop hairpins, has been implicated as a multifaceted source of error. It is also reported that these non-canonical secondary structures show an increased propensity for oxidative damage; for example, the guanine in the loop region of a (CAG)n stem-loop hairpin is a hotspot for oxidation. Non-canonical DNA secondary structures have been further shown to impact the action of enzymes in the base excision repair (BER) pathway, by which these oxidatively damaged bases are removed. More specifically, there is evidence that trinucleotide repeat-containing DNA mistakenly enters long-patch BER, which can potentially lead to the incorporation of extra nucleobases by DNA polymerase.The final enzyme in the long-patch BER pathway is DNA Ligase I, which catalyzes the formation of a phosphodiester bond to seal a nick site. When extra nucleotides have been added during an erroneous long-patch BER process, the action of DNA ligase I may expand the repeat tract by incorporating these additional bases into duplex DNA. In this study, DNA constructs containing (CAG)n hairpins at various distances from a nick site are used to investigate the ability of human DNA Ligase I to ligate substrates containing non-canonical secondary structure back into duplex DNA.

Activity of DNA ligase on substrates containing non-canonical structures

247th National Meeting (2014)

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