Proofreading and Secondary Structure Processing Determine the Orientation Dependence of CAG·CTG Trinucleotide Repeat Instability in Escherichia coli

Expanded CAG.CTG trinucleotide repeat tracts are associated with several human inherited diseases including Huntington's disease, myotonic dystrophy and spinocerebellar ataxias. Here we describe a new model system to investigate repeat instability in the Escherichia coli chromosome. Using this system, we reveal patterns of deletion instability consistent with secondary structure formation in vivo and address the molecular basis of orientation-dependent instability. We demonstrate that the orientation-dependence of CAG.CTG trinucleotide repeat deletion is determined by the proofreading subunit of DNA polymerase III (DnaQ) in the presence of the hairpin nuclease SbcCD (Rad50/Mre11). Our results suggest that although initiation of slippage can occur independently of CAG.CTG orientation, the folding of the intermediate affects its processing and this results in orientation-dependence. We propose that proofreading is inefficient on the CTG containing strand because of its ability to mis-fold and SbcCD contributes to processing in a manner that is dependent on proofreading and repeat tract orientation. Furthermore, we demonstrate that transcription and recombination do not influence instability in this system.

Key Words: Genetic disease, Genetic instability, Proofreading, Trinucleotide repeat

This article has been cited by other articles:

				E. Delagoutte, G. M. Goellner, J. Guo, G. Baldacci, and C. T. McMurray Single-stranded DNA-binding Protein in Vitro Eliminates the Orientation-dependent Impediment to Polymerase Passage on CAG/CTG Repeats J. Biol. Chem., May 9, 2008; 283(19): 13341 - 13356. [Abstract] [Full Text] [PDF]

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