Analysis of triplet repeat replication by two-dimensional gel electrophoresis.

Abstract

Expansions of triplet repeats are responsible for more than 15 hereditary neurological disorders in humans. Triplet repeats are fairly stable when the number of elementary units is under approx 30, but become polymorphic in length with a clear bias for expansions when this threshold is exceeded. This results in the rapid addition of hundreds or even thousands of extra repeats and, ultimately, disease. The mechanisms of triplet repeat expansions are not yet understood. The role of several genetic processes, including replication, recombination, and repair, was suggested. However, given the swift accumulation of extra DNA material, DNA replication seems to be an intuitive candidate for generating expansions. Numerous data point to the aberrant replication of triplet repeats as a cause of triplet repeat expansions. Direct experimental proof of aberrant replication through triplet repeats was lacking. This encouraged us to study the mode of replication fork progression through triplet repeats in vivo. We analyzed the effects of triplet repeats on replication of bacterial or yeast plasmids using an approach called two-dimensional neutral/neutral gel electrophoresis of replication intermediates. This technique, originally developed for mapping replication origins, is also instrumental in defining replication pause sites. Using this technique, we were able to unambiguously demonstrate that expandable triplet repeats attenuate replication fork progression in vivo and get some insights into the mechanisms of repeat expansions.