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Title: Investigations into site-specifies labile modifications in the DNA of Streptomyces lividans
Author: Ray, T. D.
Awarding Body: University College of Swansea
Current Institution: Swansea University
Date of Award: 1995
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The DNA of Streptomyces lividans 66 contains an unidentified site-specific modification. The modification occurs closely opposed on both DNA strands within a G+C rich sequence (P. Dyson, unpub.). A consequence of the modification is that chromosomal and plasmid DNA undergo cleavage at the site of modification when electrophoresed in standard buffers. Conditions refractive to this degradation were elucidated which employed a Hepes based electrophoresis buffer. This subsequently allowed Tris to be identified as the buffer component upon which site-specific cleavage was dependent. Time course studies demonstrated that the nucleolytic component was formed at the anode during electrophoresis and was in sufficient quantities to cause maximal DNA cleavage after four minutes activation. Quantification of the species was achieved using reduced methyl viologen in a spectrophotometric assay and found to be approximately 160μM after four minutes and 240μM after ten minutes activation. The cleavage reaction itself proceeds slowly suggesting a stable reactive species. Both DNA cleavage and spectrophotometric assays agreed with this by finding the half life of the oxidant to be 14.5 hours. The addition of reducing agents to the activated buffer inhibited DNA cleavage in a redox-potential dependent manner when non-sulphurous compounds were used. Compounds with sulphur containing moieties were generally more effective at inhibition and could be ordered in relation to their pKSH. Investigations to identify the oxidant responsible for DNA cleavage were consistent with it being a peracid species formed by oxidation of one or more hydroxymethyl groups of Tris. Preliminary studies into the sequence requirement for modification in vivo suggested that regions upstream of the putative modification site were necessary for maximal modification.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available