| Trinucleotide repeat (TNR) alleles undergo high frequency mutagenesis to cause at least 15 neurodegenerative disorders. While these diseases are devastating and often fatal, they show several fascinating genetic properties. Importantly, repeats of the sequence CNG (where N is any nucleotide) or GAA are unstable, while others are genetically inert. In 1995, it was shown that CNG and GAA repeats form can hairpins and other aberrant structures in vitro. As such, DNA replication, DNA repair, recombination and transcription are all potential mutagenic culprits, since each process exposes single-strand DNA, and error-prone metabolism of TNR DNA could allow hairpin formation and TNR mutations.;While expansions are the predominant TNR mutation in human patients, the reverse process of TNR contraction is actually favored in the normal population and model organisms. Since TNR contractions might be exploited as a means to shorten expanded alleles, I used a sensitive and quantitative shuttle vector assay to investigate the mechanism of TNR contractions in cultured human and simian cells.;Studies in immortalized human astrocytes demonstrated CAG•CTG contractions with frequencies approaching 2%. These contractions exhibited an apparent threshold between 25-33 repeats. Over this narrow range of allele lengths, contraction frequency increased 530%. This threshold-like behavior was also recapitulated in COS-1 cells.;Moreover, hairpin-forming capacity of the TNR sequence stimulated contractions by 300-1000% in SVG-A astrocytes and 293T kidney cells. Furthermore, contractions in SVG-A cells were sensitive to the direction of DNA replication. Contractions were favored >10-fold when CAG repeats comprised the template for lagging strand synthesis. Surprisingly, the presence of two interruptions in a 33-repeat tract stimulated contractions three-fold compared to a pure repeat, and 69% of the contractions retained the interruption. As stable alleles of some TNR-containing genes often contain interruptions, perhaps interruptions help prevent expansions by stimulating contraction of alleles near the threshold.;Finally, studies in several cell lines suggest that the mismatch repair proteins MLH1 and MSH2 play no detectable role in promoting or preventing TNR contractions near the threshold. |
