105 Activity of DNA ligase on substrates containing non-canonical structures

Abstract

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 (Kovtun et al., 2008). While the molecular mechanism of this expansion is unknown, the ability of trinucleotide repeat sequences to form non-canonical secondary structures, such as hairpins, has been implicated as a multifaceted source of error (Gacy et al., 1995). Non-canonical DNA secondary structures have been shown to impact the action of enzymes in the base excision repair (BER) pathway, by which 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 (Jarem et al., 2011). The final enzyme in the BER pathway is DNA Ligase, which catalyses the formation of a phosphodiester bond to seal a nick site (Taylor et al., 2011). When extra nucleotides have been added during an erroneous long-patch BER process, the action of DNA ligase 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 DNA Ligase to ligate substrates containing non-canonical secondary structure back into duplex DNA.