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.

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