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dc.contributor.advisorEl Karoui, Meriem
dc.contributor.advisorGranneman, Sander
dc.contributor.authorKalita, Irina
dc.date.accessioned2022-08-16T11:13:42Z
dc.date.available2022-08-16T11:13:42Z
dc.date.issued2022-08-16
dc.identifier.urihttps://hdl.handle.net/1842/39309
dc.identifier.urihttp://dx.doi.org/10.7488/era/2560
dc.description.abstractTo preserve genome integrity, all living organisms have developed strategies to respond to chromosomal damage. One such response is the repair of double-strand breaks (DSBs), one of the most toxic forms of DNA damage. In Escherichia coli, DSBs are repaired via a homologous recombination pathway, which is initiated by a multifunctional enzyme, RecBCD. Primarily, RecBCD detects a double-strand end and degrades the chromosome until it encounters a short DNA motif known as a Chi site. Upon Chi site recognition, RecBCD activity is modified, resulting in the formation of a RecA-coated filament which is responsible for homology search. Because of its important function in DSB repair, RecBCD is essential for accurate chromosome maintenance. On the other hand, overproduction of the enzyme has been observed to reduce DNA repair ability of bacterial cells. This double-natured phenomenon led to the idea that RecBCD expression needs to be maintained within an optimal range. Being mainly focused on the RecB subunit of the RecBCD complex, this work has addressed the following questions: how abundant is the enzyme within cells and how variable is its expression? Is there a regulation mechanism that maintains RecBCD expression at the optimal level? Using single-molecule microscopy techniques (smFISH and HaloTag-labelling), the low abundance of RecB subunits (∼ 5 molecules per cell) was confirmed and weak recBCD transcription (less than one mRNAs per cell) was further established. With a simple stochastic model of gene expression, it was shown that while transcriptional outputs are noisy, cell-to-cell protein variability is lower than expected for an unregulated gene. Secondly, RecB mRNA and protein production was shown to be uncoupled in DNA-damage conditions: a decrease in recB transcription was compensated with more efficient translation upon DSB induction. Taken together, these observations point towards a post-transcriptional regulation mechanism. A post-transcriptional regulator, Hfq, was shown to bind recBCD mRNAs and the downregulation of RecB expression by Hfq was established in vivo. A molecular mechanism of RecB production whereby Hfq inhibits RecB translation was summarized in a stochastic model and further tested with Hfq mutants. A less effective noise reduction was detected when the Hfq-mediated regulation was perturbed. This is the first evidence of Hfq involvement in suppression of protein fluctuations in bacterial cells. Additionally, disruption of the uncovered mechanism was shown to reduce DNA repair capacity of E. coli, suggesting a potential phenotypical advantage of limitation of RecB numbers. All in all, a novel Hfq-mediated regulation mechanism of RecB expression is presented in this thesis. Protein noise reduction and protection against the toxic consequences of high RecBCD numbers are proposed as potential phenotypical advantages of the uncovered fine-tuning mechanism.en
dc.contributor.sponsorThe Darwin Trust of Edinburghen
dc.language.isoenen
dc.publisherThe University of Edinburghen
dc.relation.hasversionURL: https://gitlab.com/ikalita/cellsegmentation\_mek.en
dc.relation.hasversionURL: https://gitlab.com/ikalita/spotdetection\_spatzcells.en
dc.subjectEscherichia colien
dc.subjectE. colien
dc.subjectDNA repair pathwayen
dc.subjectRecBCD enzymeen
dc.subjectRecB protein levelsen
dc.subjectHfq-mediated regulationen
dc.titleRegulation of RecBCD expression in Escherichia colien
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen
dc.rights.embargodate2023-08-16en
dcterms.accessRightsRestricted Accessen


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