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Title: Mapping replication fork blockages in a protein-rich environment
Author: Moon, Peter
ISNI:       0000 0004 2682 8527
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2009
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The structure specificity of PriA is manifested as a preference for branched DNA with a single stranded leading strand positioned at the branch point.  The nucleoid associated protein HU displays similar structure specificity.  Furthermore, HU is present in vast excess in the cell in compassion to PriA.  My results show that only a moderate excess of HU is enough to inhibit PriA function in vitro suggesting DNA-protein complexes may cause a serious obstacle to replication restart. The Escherichia coli chromosome is coated with a multitude of DNA-binding proteins.  The second part of my thesis used controllable site-specific roadblocks to replication to better understand how the replication machinery copes with protein-DNA complexes.  These roadblocks consisted of thirty four tandom lac repressor-operator complexes.  Using anti-SSB antibodies to immunoprecipitate DNA in a strain containing lacO-Lacl complexes then hybridising the DNA to specially designed microarrays (ChlP-on-chip) it was possible to map the consequence of when a replication fork encounters a protein-DNA roadblock.  It has been suggested that Rep and UvrD may play a role in the removal of protein roadblocks ahead of the replication fork.  Unlike the single mutants, Δrep ΔuvrD double mutants are inviable suggesting they share an essential role.  Unpublished work by the Lloyd group has shown that mutations in the β subunit of RNA polymerase, termed rpo* mutations, suppress the lethality of Δrep ΔuvrD mutations.  Using this information it was possible to create a Δrep ΔuvrD rpo* mutant containing the 34 lacO-Lacl complexes.  Furthermore, I was able to use ChlP-on-chip to investigate Δrep, ΔuvrD and Δrep ΔuvrD mutant strains to suggest possible roles of Rep and UvrD in vivo.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Escherichia coli