Title: Characterization of Dna ∆96-120: The role of domain II of DnaA in replication initiation
Abstract: A 25 amino acid deletion in domain II of the Escherichia coli replication initiator protein DnaA was characterized in order to further understand the mechanism by which DnaA serves as a master regulator protein of DNA replication initiation in bacteria. DnaA plays a major role in the assembly of the replisome during DNA replication and the 4 domains of DnaA bas been extensively studied with the exception of domain II which was initially characterized as a non-essential flexible linker region. Mutant dnaAtJ. 96.t2o was found to serve as a suppressor of mutant seq A phenotypes that have been shown to cause excessive over-initiation of replication (Sutera and Lovett, 2006). This suggests that this 25 amino acid region of domain II is somehow involved in the mechanisms of DNA replication initiation. As DnaA is known to interact with DiaA (DnaA-initiatorassociating factor), DNA at the origin, and DnaB and DnaC in the assembly of the replication machinery, these interactions were of particular interest in determining why the deletion of residues 96-I 20 of domain TJ mitigates such over-replication. Through colony morphology studies, DNA damage assays, and flow cytometric analysis, it was confirmed that dnaAtJ. 96.12o results in under-initiation of DNA replication and causes suppression of the mutant seqA phenotype. Additionally, DNA damage survival assays and flow cytomctry suggest that dnaA and diaA are epistatic. Through protein-protein interaction experiments it was determined that the deletion of 25 amino acids of domain II did not inhibit binding of DnaA to DiaA. Flow cytometry and DNA damage survival assays suggest that dnaAtJ. 96.t2o impacts DNA replication by causing under-initiation and that the non-essential domain II plays a regulatory role in initiation of replication.
Publication Year: 2009
Publication Date: 2009-01-01
Language: en
Type: dissertation
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