DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli

Abstract

The influence of mutations in the 3' to 5' exonucleolytic proofreading ε-subunit of Escherichia coil DNA polymerase III on the genetic instabilities of the CGG·CCG and the CTG·CAG repeats that cause human hereditary neurological diseases was investigated. The dnaQ49(ts) and the mutD5 mutations destabilize the CGG·CCG repeats. The distributions of the deletion products indicate that slipped structures containing a small number of repeats in the loop mediate the deletion process. The CTG·CAG repeats were destabilized by the dnaQ49(ts) mutation by a process mediated by long hairpin loop structures (≥5 repeats). The mutD5 mutator strain stabilized the (CTG·CAG)175 tract, which contained two interruptions. Since the mutD5 mutator strain has a saturated mismatch repair system, the stabilization is probably an indirect effect of the nonfunctional mismatch repair system in these strains. Shorter uninterrupted tracts expand readily in the mutD5 strain, presumably due to the greater stability of long CTG·CAG tracts (> 100 repeats) in this strain. When parallel studies were conducted in minimal medium, where the mutD5 strain is defective in exonucleolytic proofreading but has a functional MMR system, both CTG·CAG and CGG·CCG repeats were destabilized, showing that the proofreading activity is essential for maintaining the integrity of TRS tracts. Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that reduce the fidelity of replication.