PAUSING OF DNA-SYNTHESIS IN-VITRO AT SPECIFIC LOCI IN CTG AND CGG TRIPLET REPEATS FROM HUMAN HEREDITARY-DISEASE GENES

Several human hereditary neuromuscular disease genes are associated wi th the expansion of CTG or CGG triplet repeats. The DNA syntheses of C TG triplets ranging from 17 to 180 and CGG repeats from 9 to 160 repea ts in length were studied in vitro. Primer extensions using the Klenow fragment of DNA polymerase I, the modified T7 DNA polymerase (Sequena se), or the human DNA polymerase beta paused strongly at specific loci in the CTG repeats. The pausings were abolished by heating at 70 degr ees C. As the length of the triplet repeats in duplex DNA, but not in single-stranded DNA, was increased, the magnitude of pausings increase d. The location of the pause sites was determined by the distance betw een the site of primer hybridization and the beginning of the triplet repeats. CGG triplet repeats also showed similar, but not identical, p atterns of pausings. These results indicate that appropriate lengths o f the triplets adopt a non-B conformation(s) that blocks DNA polymeras e progression; the resultant idling polymerase may catalyze slippages to give expanded sequences and hence provide the molecular basis for t his non-Mendelian genetic process. These mechanisms, if present in hum an cells, may be related to the etiology of certain neuromuscular dise ases such as myotonic dystrophy and Fragile X syndrome.