MISMATCHED NUCLEOTIDES MAY FACILITATE EXPANSION OF TRINUCLEOTIDE REPEATS IN GENETIC-DISEASES

We have studied the contribution of mismatch sequences to the trinucle otide repeat expansion that causes hereditary diseases. Using an oligo nucleotide duplex, (CAG)(5)/(CTG)(5), as a template-primer, DNA synthe sis was carried out using either Escherichia coli DNA polymerase I (Kl enow fragment) or human immunodeficiency virus type I reverse transcri ptase (HIV-RT). Both enzymes expanded the repeat sequence longer than 27 nucleotides (nt), beyond the maximum length expected from the templ ate size, The expansion was observed under conditions in which extensi on occurs either in both strands or in one strand. In contrast, with a nother template-primer that contains a non-repetitive flanking sequenc e 5'-upstream of the repetitive sequence, the reaction products were n ot extended beyond the template size (45 nt) by these DNA polymerases. We then used mismatched template-primers, in which either 1, 2 or 6 n on-complementary nucleotides were introduced to the repeat sequence th at is flanked by a non-repetitive sequence. In this case, primers were efficiently expanded over the expected length of 45 nt, in a mismatch -dependent manner. One of the primers with six mismatches extended as long as 72 nt. These results imply that the misincorporation of non-co mplementary deoxyribonucleoside monophosphates (dNMPs) into the repeat sequence makes double-stranded DNA unstable and triggers the slippage and expansion of trinucleotide repeats by forming loops or hairpin st ructures during DNA synthesis.