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Title: CTG trinucleotide repeat instability in Escherichia coli
Author: Schmidt, Kristina H.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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In order to identify cellular factors that affect trinucleotide repeat stability, changes in the length of a (CTG)43 repeat were studied over 140 generations in wild-type Escherichia coli and in strains that are deficient in post-replicative mismatch repair, secondary structure repair and homologous recombination. It is shown that (CTG)43 inserted into pUC18 expands and contracts in wild-type E. coli in an orientation-dependent manner that is unaffected by transcription. In cells deficient in post-replicative mismatch repair (CTG)43 repeat instability is greater than in wild-type cells but orientation-independent. The observation of single trinucleotide insertions and deletions in these mutator mutants indicates that replication slippages of 3 bp occur in vivo leading to repeat expansion and contraction if left unrepaired. Compared to wild-type cells large deletions are reduced in these mutator mutants, but only if the CTG sequence serves as the lagging strand. Based on the opposing effects of mismatch repair a model is proposed in which orientation-dependent CTG repeat instability in mismatch repair proficient cells is caused by the repair of 3-bp slippages. This leads to the creation of larger deletions during repair synthesis due to the formation of unusual secondary structures by the CTG sequence on the lagging strand. Mutations in the recA and sbcCD genes do not affect the stability of plasmid-borne CTG repeats. Similarly the viability of recA-deficient strains carrying chromosomal insertions of (CTG)25 and (CTG)43 suggests that, unlike long palindromes, these trinucleotide repeats are not substrates for the structure-directed nuclease complex SbcCD or, alternatively, they do not form secondary structures frequently enough to cause lethality in recA-deficient hosts. In contrast, a mutation in the recG gene, also involved in homologous recombination, severely destabilises the (CTG)43 repeat in a strongly orientation-dependent manner that exceeds all other tested mutants. Possible explanations for this observation are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available