The influence of negative superhelical density on the genetic instabilities of long GAA·TTC, CGG·CCG, and CTG·CAG repeat sequences was studied in vivo in topologically constrained plasmids in Escherichia coli. These repeat tracts are involved in the etiologies of Friedreich ataxia, fragile X syndrome, and myotonic dystrophy type 1, respectively. The capacity of these DNA tracts to undergo deletions-expansions was explored with three genetic-biochemical approaches including first, the utilization of topoisomerase I and/or DNA gyrase mutants, second, the specific inhibition of DNA gyrase by novobiocin, and third, the genetic removal of the HU protein, thus lowering the negative supercoil density (-σ). All three strategies revealed that higher -σ in vivo enhanced the formation of deleted repeat sequences. The effects were most pronounced for the Friedreich ataxia and the fragile X triplet repeat sequences. Higher levels of -σ stabilize non-B DNA conformations (i.e. triplexes, sticky DNA, flexible and writhed DNA, slipped structures) at appropriate repeat tracts; also, numerous prior genetic instability investigations invoke a role for these structures in promoting the slip-page of the DNA complementary strands. Thus, we propose that the in vivo modulation of the DNA structure, localized to the repeat tracts, is responsible for these behaviors. Presuming that these interrelationships are also found in humans, dynamic alterations in the chromosomal nuclear matrix may modulate the -σ of certain DNA regions and, thus, stabilize/destabilize certain non-B conformations which regulate the genetic expansions-deletions responsible for the diseases. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.