Regulation of RecBCD expression in Escherichia coli
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Date
16/08/2022Item status
Restricted AccessEmbargo end date
16/08/2023Author
Kalita, Irina
Metadata
Abstract
To 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.