dc.contributor.advisor | Leach, David | en |
dc.contributor.author | Jackson, Adam | en |
dc.date.accessioned | 2011-02-08T15:32:09Z | |
dc.date.available | 2011-02-08T15:32:09Z | |
dc.date.issued | 2010 | |
dc.identifier.uri | http://hdl.handle.net/1842/4782 | |
dc.description.abstract | A trinucleotide repeat (TNR) is a 3 base pair (bp) DNA sequence tandemly
repeated in an array. In humans, TNR sequences have been found to be
associated with at least 14 severe neurological diseases including Huntington
disease, myotonic dystrophy and several of the spinocerebellar ataxias. Such
diseases are caused by an expansion of the repeat sequence beyond a threshold
length and are characterized by non-Mendelian patterns of inheritance which
lead to genetic anticipation. Although the mechanism of the genetic instability
in these arrays is not yet fully understood, various models have been suggested
based on the in vitro observation that TNR sequences can form secondary
structures such as pseudo-hairpins.
In order to investigate the mechanisms responsible for instability of TNR
sequences, a study was carried out on Escherichia coli cells in which TNR arrays
had been integrated into the chromosomal lacZ gene. This genetic assay was
used to identify proteins and pathways involved in deletion and/or expansion
instability.
Deletion instability was clearly dependent on orientation of the TNR
sequence relative to the origin of replication. Interestingly, it was found that
expansion instability is not dependent on the orientation of the repeat array
relative to the origin of replication.
The replication fork reversal pathway and the RecFOR mediated gap
repair pathway were found to have no statistically significant influence on the
instability of TNR arrays. However, the protein UvrD was found to affect the
deletion instability of TNR sequences.
The roles of key helicase genes were investigated for their effects on
instability of chromosomal CTG•CAG repeats. Mutation of the rep gene
increased deletion in the CTG leading-strand orientation of the repeat array, and
expansion in both orientations - destabilizing the TNR array. RecQ helicase was
found to have a significant effect on TNR instability in the orientation in which
CAG repeats were present on the leading-strand relative to the origin of
replication. Mutation of the recQ gene severely limited the number of expansion
events in this orientation, whilst having no effect on deletions. This dependence
of expansions on RecQ was lost in a rep mutant strain. In a rep mutant
expansions were shown to be partially dependent on the DinG helicase.
All together, these results suggest a model of TNR instability in which
expansions are due to events occurring at either the leading or lagging strand of
an arrested replication fork, facilitated by helicase action. The identity of the
helicase implicated is determined by the nature of the arrest. | en |
dc.contributor.sponsor | Medical Research Council (MRC) | en |
dc.language.iso | en | |
dc.publisher | The University of Edinburgh | en |
dc.subject | trinucleotide repeat | en |
dc.subject | TNR | en |
dc.subject | genetic instability | en |
dc.subject | pseudo-hairpins | en |
dc.subject | genetic assay | en |
dc.subject | UvrD | en |
dc.subject | arrested replication fork | en |
dc.subject | helicase | en |
dc.title | Effect of helicases on the instability of CTG・CAG trinucleotide repeat arrays in the escherichia coli chromosome | en |
dc.type | Thesis or Dissertation | en |
dc.type.qualificationlevel | Doctoral | en |
dc.type.qualificationname | PhD Doctor of Philosophy | en |