Mechanisms of expansion and deletion of triplet repeat sequences that cause human hereditary neurological diseases.

摘要

The expansion of triplet repeat sequences has been identified as the causative mutation of a number of human hereditary neurodegenerative diseases. Replication-based strand slippage was previously suggested to result in triplet repeat expansion. Using the genetically tractable Escherichia coli, we investigated the role replication and repair in the genetic instability of triplet repeats. We studied the replication properties of DNA substrates containing the CTG·CAG, CGG·CCG and GAA·TTC repeats in an in vivo filamentous phagemid replication system that lacked a discontinuous lagging strand. The repeat tracts were substantially deleted when the CTG, CGG and GAA repeats were the templates for continuous rolling circle replication from the filamentous phage f1 origin. In contrast, the tracts were more stable when CAG, CCG and TTC comprised the rolling circle template. Therefore we concluded that the triplet repeat instabilities arose due to the formation of hairpin-loops in the rolling circle template. This conclusion was based on previous proposals that the CTG, CGG and GAA repeats have higher propensities to form secondary structures their complementary CAG, CCG or TTC repeats. Since rolling circle replication has been established as an in vitro model system for leading strand synthesis, we conclude that triplet repeat instabilities can occur due to the formation of secondary structures on the leading strand of DNA replication. Previous work had shown that repair processes had an influence on the genetic instabilities of triplet repeats. We analyzed the expansion and deletion of the CGG·CCG and CTG·CAG repeats in strains of E. coli that had mutations in the proofreading 3′ to 5′ exoncleolytic ϵ-subunit of DNA polymerase III. We observed that the dnaQ49ts and mutD5 mutations destabilized the CGG·CCG repeats. The deletion product distributions indicated that slipped structures were the intermediates of the deletion process. The dnaQ49ts mutation destabilized the CTG·CAG repeats via intermediates containing longer hairpin-loops (>5 repeats). Shorter uninterrupted CTG·CAG tracts expand in the mutD5 mutator strain. Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that affect the fidelity of DNA replication.