Hairpin formation during DNA synthesis primer realignment in vitro in triplet repeat sequences from human hereditary disease genes.

Abstract

Genetic expansion of DNA triplet repeat sequences (TRS) found in neurogenetic disorders may be due to abnormal DNA replication. We have previously observed strong DNA synthesis pausings at specific loci within the long tracts (> approximately 70 repeats) of CTG.CAG and CGG.CCG as well as GTC.GAC by primer extensions in vitro using DNA polymerases (the Klenow fragment of Escherichia coli DNA polymerase I, the modified T7 DNA polymerase (Sequenase), and human DNA polymerase beta). Herein, we have isolated and analyzed the products of stalled synthesis found at approximately 30-40 triplets from the beginning of the TRS. DNA sequence analyses revealed that the stalled products contained short tracts of homogeneous TRS (6-12 repeats) in the middle of the sequence corresponding to the flanking region of the primer-template sequence. The sequence at the 3'-side terminated at the end of the primer, indicating that the primer molecule had served as a template. In addition, chemical probe and polyacrylamide gel electrophoretic analyses revealed that the stalled products existed in hairpin structures. We postulate that these products of the DNA polymerases are caused by the existence of an unusual DNA conformation(s) within the TRS, during the in vitro DNA synthesis, enhancing the DNA slippages and the hairpin formations in the TRS due to primer realignment. The consequence of these steps is DNA synthesis to the end of the primer and termination. Primer realignment including hairpin formation may play an important intermediate role in the replication of TRS in vivo to elicit genetic expansions.