Genetic Instabilities in (CTG super(.)CAG) Repeats Occur by Recombination

• Published in: The Journal of biological chemistry 1999-08, Vol.274 (33), p.23468-23479
• Main Authors: Jakupciak, J P, Wells, R D
• Format: Article
• Language: English
• ISSN: 0021-9258
• DOI: 10.1074/jbc.274.33.23468

The expansion of triplet repeat sequences (TRS) associated with hereditary neurological diseases is believed from prior studies to be due to DNA replication. This report demonstrates that the expansion of (CTG super(.)CAG)n in vivo also occurs by homologous recombination as shown by biochemical and genetic studies. A two-plasmid recombination system was established in Escherichia coli with derivatives of pUC19 (harboring the ampicillin resistance gene) and pACYC184 (harboring the tetracycline resistance gene). The derivatives contained various triplet repeat inserts ((CTG super(.)CAG), (CGG super(.)CCG), (GAA super(.)TTC), (GTC super(.)GAC), and (GTG super(.)CAC)) of different lengths, orientations, and extents of interruptions and a control non-repetitive sequence. The availability of the two drug resistance genes and of several unique restriction sites on the plasmids enabled rigorous genetic and biochemical analyses. The requirements for recombination at the TRS include repeat lengths >30 the presence of CTG super(.)CAG on both plasmids, and recA and recBC. Sequence analyses on a number of DNA products isolated from individual colonies directly demonstrated the crossing-over and expansion of the homologous CTG super(.)CAG regions. Furthermore, inversion products of the type [(CTG) sub(13)(CAG) sub(67)] super(.)[(CTG) sub(67)(CAG) sub(13) ] were isolated as the apparent result of "illegitimate" recombination events on intrahelical pseudoknots. This work establishes the relationships between CTG super(.)CAG sequences, multiple fold expansions, genetic recombination, formation of new recombinant DNA products, and the presence of both drug resistance genes. Thus, if these reactions occur in humans, unequal crossing- over or gene conversion may also contribute to the expansions responsible for anticipation associated with several hereditary neurological syndromes.