Quantitative Studies Of The Dynamics Of Prokaryotic DNA Damage Response

Cellular DNA is frequently attacked by various chemical or physical agents that cause lesions. DNA lesions can lead to genetic instability. To cope with such crisis, cells have evolved an elaborate system which responsible for cell cycle control, damage repair and, in eukaryote, apoptosis. Some DNA repair mechanism can even induce mutagenesis so that the offspring may adapt to the new environment. In order to gain a grater probability to survive, all these tasks have to be done cooperatively. The SOS response of Escherichia coli is a classic model system for studying DNA damage repair response and there has been plenty of quantitative datas about it. With the mathematical models describing the SOS response, I intent to analysis these datas integratively and explore the evolutionary advantage of DNA damage response mechanisms.For the first time, a stochastic model is built to describe the SOS response. The model can explain the oscillatory modulation of inducing signal for SOS response in single cell. We argue that single cell shows oscillatory modulation because a limited number of replication forks occasionally encounter DNA lesions and trigger the inducing signal. By fitting the model parameters and stability analysis, we verify that the triggering time interval is not random but govern by the dynamic properties of the system.In order to further testify our model, we design a molecular biological experiment to quantitatively measure the expression level of the mutagenesis protein UmuD by immunoblot. We obtain an antibody with proper affinity to both UmuD and UmuD'so that the in vivo level of these two proteins can be quantified simultaneously. As a result, we manage to estimate the number of protein molecules per single cell. We find that with the increase of damage, the expression profile of UmuD/UmuD'changes significantly.In order to explain such changes, we modify the model to include the fact that DNA damage causes the cell growth slowing down. By fitting the UmuD/UmuD'dynamic curve to experimental data, we find that the SOS mutagenesis events mainly take place after most of the lesions have been removed. It means that SOS mutagenesis is used mainly as a strategy for evolutionary adaptation, but not assisting DNA replication recovering after DNA damage.For application study, we use the elements from SOS response system to construct a push-on-push-off switch gene circuit. The circuit can sense Ultra-Violet light (UV) irradiation as input signal. Under intermittent irradiation, two reporter genes are to be induced alternately. The circuit can be divided into two associated parts: a bistable switch and a NOR gate. The NOR gate is controlled by the bistable switch and the input signal. On the other hand, the output of the NOR gate can switch the bistable state from one to the other. A mathematical model is built to verify the feasibility of the design. Thus far, we have constructed a series of NOR gates and bistable switches as candidates for final combination.