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 with the expansion of CTG or CGG triplet repeats. The DNA syntheses of CTG triplets ranging from 17 to 180 and CGG repeats from 9 to 160 repeats in length were studied in vitro. Primer extensions using the Klenow fragment of DNA polymerase I, the modified T7 DNA polymerase (Sequenase), or the human DNA polymerase beta paused strongly at specific loci in the CTG repeats. The pausings were abolished by heating at 70 degrees C. As the length of the triplet repeats in duplex DNA, but not in single-stranded DNA, was increased, the magnitude of pausing increased. The location of the pause sites was determined by the distance between the site of primer hybridization and the beginning of the triplet repeats. CGG triplet repeats also showed similar, but not identical, patterns of pausings. These results indicate that appropriate lengths of the triplets adopt a non-B conformation(s) that blocks DNA polymerase progression; the resultant idling polymerase may catalyze slippages to give expanded sequences and hence provide the molecular basis for this non-Mendelian genetic process. These mechanisms, if present in human cells, may be related to the etiology of certain neuromuscular diseases such as myotonic dystrophy and Fragile X syndrome.
Kang
S
S
Institute of Biosciences and Technology, Texas A & M University, Texas Medical Center, Houston 77030, USA.
Ohshima
K
K
Shimizu
M
M
Amirhaeri
S
S
Wells
R D
RD
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D018598:Minisatellite Repeats; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009468:Neuromuscular Diseases / Q000235:genetics* / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature; D018911:Trinucleotide Repeats
D017931:DNA Primers; D004247:DNA; D004259:DNA-Directed DNA Polymerase