Effects of temperature, Mg2+ concentration and mismatches on triplet-repeat expansion during DNA replication in vitro.
The human genome contains many simple tandem repeats that are widely dispersed and highly polymorphic. At least one group of simple tandem repeats, the DNA trinucleotide repeats, can dramaticallyexpand in size during transmission from one generation to the next to cause disease by a process known as dynamic mutation. We investigated the ability of trinucleotide repeats AAT and CAG to expand in size during DNA replication using a minimal in vitro system composed of the repeat tract, with and without unique flanking sequences, and DNA polymerase. Varying Mg2+concentration and temperature gave dramatic expansions of repeat size during DNA replication in vitro. Expansions of up to 1000-fold were observed. Mismatches partially stabilized the repeat tracts against expansion. Expansions were only detected when the primer was complementary to the repeat tract rather than the flanking sequence. The results imply that cellular environment and whether the growing strand contains a nick or gap are important factors for the expansion process in vivo.
D020137:Base Pair Mismatch; D002413:Cations, Divalent; D004261:DNA Replication / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008274:Magnesium; D008278:Magnesium Sulfate / Q000494:pharmacology; D013696:Temperature; D018911:Trinucleotide Repeats