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Title: Detection of cruciform DNA in vivo
Author: Allers, Thorsten
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1993
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This work describes an internally controlled, non-invasive assay for cruciform DNA in vivo, which relies on the inhibition of methylation at target sites that are in regions of unusual DNA secondary structure. If a palindrome with a central GATC target site for Dam methylase extrudes as a cruciform structure, then the GATC sequence will be located in a single-stranded loop and will consequently not be modified. The centre of of a 476 bp perfect palindrome located in a bacteriophage λ derivative is shown to adopt a methylation-resistant DNA structure that is consistent with cruciform formation in vivo. Changes to the central base pairs are found to affect methylation inhibition, arguing that this extrusion occurs by a centre-dependent pathway. Using kinetic modelling, it is estimated that the palindrome centres examined are inaccessible to Dam methylase for between 35% and 88% of the λ lytic lifecycle. A palindrome with an asymmetric insertion of 10 bp located in a bacteriophage λ derivative is found to exhibit unexpectedly high levels of methylation inhibition at a central GATC target site. In contrast, the λ phage carrying this inverted repeat shows a plating behaviour that is consistent with previous observations, which indicate that central asymmetry permits efficient replication by disfavouring cruciform extrusion in vivo. Furthermore, the viability (as measured by plaque areas on an Escherichia coli sbcC lawn) of the λ vectors carrying perfect palindromes is not commensurate with the degree of methylation inhibition at their palindrome centres in vivo. An attempt is made to reconcile these disparate results within the framework of current hypotheses for palindrome-mediated replicon inviability.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available