Several human hereditary neurological and neurodegenerative disease genes are associated with the expansion of CTG repeats. Here we show that the frequency of genetic expansions or deletions in Escherichia coli depends on the direction of replication. Large expansions occur predominantly when the CTGs are in the leading strand template rather than the lagging strand. However, deletions are more prominent when the CTGs are in the opposite orientation. Most deletions generated products of defined size classes. Strand slippage coupled with non-classical DNA structures may account for these observations and relate to expansion-deletion mechanisms in eukaryotic chromosomes for disease genes.

A family of unusual DNA structures has been discovered in segments with predominantly purines in one strand (pur.pyr sequences). These sequences are overrepresented in eukaryotic DNA and have been mapped near genes and recombination hot spots. When cloned into recombinant plasmids, many pur.pyr sequences are reactive to chemical and enzymic probes that are generally specific for single-stranded DNA. An intramolecular triplex is adopted by mirror repeats of G's and A's. Other non-B DNA structures adopted by similar sequences remain to be fully clarified but may be a family of related conformations. It is likely that these unorthodox structures play an important role in the function of the eukaryotic genome.

Friedreich ataxia (FRDA) is associated with the expansion of a GAA⅐TTC triplet repeat in the first intron of the frataxin gene, resulting in reduced levels of frataxin mRNA and protein. To investigate the mechanisms by which the intronic expansion produces its effect, GAA⅐TTC repeats of various lengths (9 to 270 triplets) were cloned in both orientations in the intron of a reporter gene. Plasmids containing these repeats were transiently transfected into COS-7 cells. A length-and orientation-dependent inhibition of reporter gene expression was observed. RNase protection and Northern blot analyses showed very low levels of mature mRNA when longer GAA repeats were transcribed, with no accumulation of primary transcript. Replication of plasmids carrying long GAA⅐TTC tracts (ϳ250 triplets) was greatly inhibited in COS-7 cells compared with plasmids carrying (GAA⅐TTC) 9 and (GAA⅐TTC) 90 . Replication inhibition was five times greater for the plasmid whose transcript contains (GAA) 230 than for the plasmid whose transcript contains (UUC) 270 . Our in vivo investigation revealed that expanded GAA⅐TTC repeats from intron I of the FRDA gene inhibit transcription rather than posttranscriptional RNA processing and also interfere with replication. The molecular basis for these effects may be the formation of non-B DNA structures.

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