title: DNA double-strand breaks induce deletion of CTG.CAG repeats in an orientation-dependent manner in Escherichia coli.
abstract: The influences of double-strand breaks (DSBs) within a triplet repeat sequence on its genetic instabilities (expansions and deletions) related to hereditary neurological diseases was investigated. Plasmids containing 43 or 70 CTG.CAG repeats or 43 CGG.CCG repeats were linearized in vitro near the center of the repeats and were transformed into parental, RecA-dependent homologous recombination-deficient, or RecBC exonuclease-deficient Escherichia coli. The resulting repair process considerably increased deletion of the repeating sequence compared to the circular DNA controls. Unexpectedly, the orientation of the insert relative to the unidirectional ColE1 origin of replication affected the amount of instability generated during the repair of the DSB. When the CTG strand was the template for lagging-strand synthesis, instability was increased, most markedly in the recA- strain. Results indicated that RecA and/or RecBC might play a role in DSB repair within the triplet repeat. Altering the length, orientation, and sequence composition of the triplet repeat suggested an important role of DNA secondary structures during repair intermediates. Hence, we hypothesize that ColE1 origin-dependent replication was involved during the repair of the DSB. A model is presented to explain the mechanisms of the observed genetic instabilities.
authors.affiliation: Institute of Biosciences and Technology, Center for Genome Research, Texas A and M University System Health Science Center, Texas Medical Center, 2121 W. Holcombe Blavd., Houston, TX 77030-3303, USA.
authors.forename: Micheal L
authors.initials: ML
authors.lastname: Hebert
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Spitz
authors.forename: Robert D
authors.initials: RD
authors.lastname: Wells
mesh_terms: D001483:Base Sequence; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D042822:Genomic Instability; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D018741:Replication Origin; D018911:Trinucleotide Repeats
chemical_list: D004269:DNA, Bacterial