In Escherichia coli, replication initiates when the DnaA-ATP protein assembles at the origin oriC. Hda protein in complex with the beta sliding clamp protein (β clamp) on DNA, functions in altering the nucleotide bound form of the replication initiator DnaA protein from DnaA-ATP to DnaA-ADP in a process termed as Regulatory Inactivation of DnaA (RIDA). DnaA also functions as a transcription factor of several genes including the aerobic ribonucleotide reductase nrdAB genes. In this study, I have exploited the cold sensitive growth phenotype due to loss of Hda function and its suppressors to understand the Hda function beyond initiation of replication. I describe the global transcriptional changes in strains lacking Hda and suppressed by two different modes. Loss of Hda function results in reduced expression of nrdAB genes, altered thiol status of the cell, SOS induction and increase in iron import due to de-repression of the Fur regulon. Strains lacking Hda function have increased requirement for the RpoH mediated heat shock response that affects the activity of NrdAB. I have shown that oversupply of β clamp results in a slow growth phenotype which is more pronounced at low temperatures. Six mutant β clamps suppress this slow growth phenotype. One of the mutant clamp that has the E202K mutation displays a hyper-Hda phenotypes such as hydroxyurea resistance and increase in nrdAB expression. These phenotypes were dependent on Hda but independent of SOS response. Finally, the slow growth phenotype due to overexpression of β clamp can be compensated by co-overproducing Hda. This leads to a model where β clamp could recruit Hda as a response to replication defects independent of SOS response.
|Commitee:||Gulick, Andrew, O'Brian, Mark, Surtees, Jennifer|
|School:||State University of New York at Buffalo|
|School Location:||United States -- New York|
|Source:||DAI-B 77/07(E), Dissertation Abstracts International|
|Keywords:||Cell death, Dna replication, Dnaa, Hda, Initiation, Oxidative stress|
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