Date of Award
2018
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Graduate Group
Cell & Molecular Biology
First Advisor
Rahul M. Kohli
Abstract
Prokaryotes possess a remarkable ability to respond to environmental stressors
using simple genetic circuits that detect signals of stress and mount an appropriate
response. The SOS pathway is an example of such a genetic circuit mediating error-prone
DNA repair in response to DNA damage. Native control over the SOS pathway is
orchestrated by the repressor-protease, LexA. Following a DNA damaging event, the
damage sensor RecA activates LexA to undergo a self-cleavage reaction that results in
LexA dissociation from SOS promoters and subsequent pathway activation. However, the
inability to decouple upstream events – DNA damage and RecA activation – from LexA
cleavage by genetic means alone has limited our ability to wholly understand how the
SOS pathway contributes to repair, mutagenesis, and bacterial survival. We sought to
overcome this limitation by designing a synthetic circuit to orthogonally control SOS
activation independent of native signals. Chapter 2 describes the design of the synthetic
circuit, in which an exogenously cleavable LexA variant was engineered by embedding a
recognition site for TEV protease into the LexA flexible linker region. TEV expression
was placed under the control of the small-molecule anhydrotetracycline (ATc),
decoupling LexA cleavage from DNA damage and RecA. We show that addition of ATc
to strains harboring our synthetic circuit permits small-molecule inducible UV resistance
and inducible mutagenesis. Further, exploiting our ability to activate SOS genes
independently of upstream events, we show that SOS pathway activation alone is
insufficient for mutagenesis, but instead demonstrate the importance of a DNA damage
nidus. In Chapter 3, as our circuit newly permits temporal separation of damage and
repair, we utilize our circuit to probe the kinetics of UV-mediated cell death and the
timeframe in which repair must occur to prevent lethality. We find delaying SOS
activation results in a rapid time-dependent loss of viability and global promoter
silencing, and that the rate of irreversible lethality is energy-dependent but protein
synthesis- and replication-independent, shedding light on the potential mechanisms of
UV-mediated cell death. Finally, in Chapter 4, we outline future uses for our synthetic
circuit to dissect the roles of the SOS pathway in repair, mutagenesis, and other
phenotypes.
Recommended Citation
Kubiak, Jeffrey, "A Synthetic Circuit For Control Of The Bacterial Dna Damage Response Without Dna Damage" (2018). Publicly Accessible Penn Dissertations. 2683.
https://repository.upenn.edu/edissertations/2683