Mechanism of in vitro expansion of long DNA repeats: effect of temperature, repeat length, repeat sequence, and DNA polymerases.
Studies of sequence repeat expansions from duplexes consisting of DNA repeat sequences greater than three bases are currently lacking. These studies are needed in order to gain a better understanding of DNA expansions in general and as a first step in understanding expansions of longer sequence repeats that have been implicated in human diseases. We have undertaken an in vitro study of tetranucleotide, hexanucleotide, and octanucleotide repeat expansions from short DNA duplexes using Taq DNA polymerase. Expansions of hexanucleotide repeats were also studied with the Klenow fragment of DNA polymerase I and with T4 DNA polymerase. Studies with Taq DNA polymerase show that expansions occur more readily as the length of the repeat sequence decreases but are generally more efficient at reaction temperatures closer to the melting point of the starting duplex. A mechanism for the observed expansions with Taq DNA polymerase is proposed that does not invoke strand slippage or DNA structure. Studies at 37 degrees C with Klenow pol I and T4 DNA polymerase indicate that the template-switching and/or strand-displacement activities of the polymerases used can play a major role in the apparent in vitro expansions of short repetitive DNA duplexes.
Tuntiwechapikul
Wirote
W
Division of Medicinal Chemistry, College of Pharmacy, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, 78712, USA.
Salazar
Miguel
M
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004256:DNA Polymerase I / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D012091:Repetitive Sequences, Nucleic Acid; D013329:Structure-Activity Relationship; D019914:Taq Polymerase / Q000378:metabolism; D013816:Thermodynamics
D004247:DNA; D019914:Taq Polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase