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Title: The mechanism of regulation of the restriction activity of the EcoKI, a type I restriction enzyme
Author: Doronina, Victoria Alexandrovna
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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The type I restriction enzyme EcoKI is an oligomeric enzyme consisting of subunits responsible for DNA target recognition (HsdS), DNA modification (HsdM) and the ATP-dependent translocation and cleavage of unmodified DNA (HsdR). It was shown in vivo and in vitro that some mutations in hsdM, which impair modification activity of EcoKI, result in a restriction-proficient modification-deficient enzyme. The survival of the mutant bacteria is dependent on the presence of functional ClpXP protease; HsdR is degraded by ClpXP in the restriction-proficient modification-deficient mutant. This degradation requires a restriction complex capable of ATP-dependent translocation and occurs before the completion of the restriction pathway. Degradation of the restriction subunit results in a phenomenon referred to as restriction alleviation (RA). If hsdR is present in high copy number, thereby increasing the level of modification-deficient EcoKI, ClpXP is unable to protect the bacterial chromosome from attack by EcoKI. A restriction-proficient modification-deficient mutant retains some residual restriction activity, which is efficient against DNA that enters the bacterium in the double-stranded form but not against DNA that enters in the single-stranded form. The level of HsdR is depleted in the cytoplasmic but not in the membrane fraction under the conditions that lead to restriction alleviation. However, compartmentalisation of EcoKI cannot explain why chromosomal DNA triggers ClpXP-dependent degradation of HsdR whereas foreign DNA does not. ClpXP protects unmodified chromosomal DNA but not foreign DNA from the resident restriction complex.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available