In order to monitor the T7 replisome fate upon encountering abasic lesion, I optimized
a single molecule flow stretching assay where the replisome encounters either abasic site or undamaged site inserted at 3.5 kilobases from the replication fork. The obtained events were categorized into three groups; bypass, restart and permanent stop. The results showed 52% bypass, 39% pause and 9% stop upon encountering the abasic lesion. The pause duration in the restart events was found to be ten times longer than the undamaged one.
Moreover, an ensemble experiment was performed, and the results were slightly consistent with regard to the bypass percentage (70%) but the stoppage percentage was significantly higher in the ensemble replication reaction (30%). Further investigations were made and it was found that the rate of the T7 replisome increases after bypassing the abasic lesion.
To inquire more about this rate switch and the difference between the single molecule and ensemble results, another unwinding experiment was performed where only gp4 (helicase) was used from the replisome. Interestingly, the rate of DNA unwinding by gp4 was similar to the rate observed after the replisome bypasses the lesion. We hypothesize that the polymerase is stalled at the abasic site and its interaction with the helicase is lost.
Consequently, the helicase and the polymerase will uncouple where the helicase continues unwinding the DNA to result in a higher observed rate after bypassing the abasic site.
Additional studies will be performed in the future to directly observe the helicase and
polymerase uncoupling upon encountering the lesion.
|Date of Award||Nov 2019|
|Original language||English (US)|
- Biological, Environmental Science and Engineering
|Supervisor||Samir Hamdan (Supervisor)|
- Abasic Lesion
- T7 Replisome
- DNA replication
- DNA damage
- Flow stretching single molecule assay
- Replication fork