Eukaryotic replication, cis-acting elements, and instability of trinucleotide repeats

Several progressive neurodegenerative diseases are caused by the expansion of an unstable triplet-repeat sequence. These sequences are somatically unstable and display tissue-specific patterns of instability. Typically, a bias towards continued expansion is seen in tissues primarily affected in these diseases. Accumulation of expansions over time is thought to underlie the tissue-specific and progressive nature of triplet-repeat diseases. Transgenic mouse models of repeat instability and analysis of somatic instability of triplet-repeats situated in various genomic locations have revealed the critical role played by the flanking sequence context in regulating repeat instability. These data support the hypothesis that of cis-acting factors that regulate repeat instability in mammals. I have used the (GAA•TTC) n sequence that causes Friedreich ataxia as a model to investigate the role of cis-acting factors in regulating triplet-repeat instability. A defined replication assay was developed and used to evaluate replication, transcription, and flanking DNA methylation as cis-acting modifiers of (GAA•TTC)n instability in mammalian cells. Replication of a (GAA•TTC)115 sequence via the SV40 origin of replication (ori) induced instability that was modified by altering the location of the ori relative to the repeat tract. The addition of transcription further destabilized the (GAA•TTC)115 sequence. The pattern of instability induced via transcription was also modified by altering the location of the ori relative to the repeat tract. Thus, location of the ori relative to the repeat tract and the location of the repeat tract within a transcriptional unit serve as cis-acting modifiers of triplet-repeat instability in mammalian cells.