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dc.contributor.advisorHamdan, Samir
dc.contributor.authorAlhudhali, Lubna F.
dc.date.accessioned2019-11-24T08:12:44Z
dc.date.available2019-11-20T00:00:00Z
dc.date.issued2019-11
dc.identifier.doi10.25781/KAUST-01928
dc.identifier.urihttp://hdl.handle.net/10754/660187
dc.description.abstractIn 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.
dc.language.isoen
dc.subjectAbasic Lesion
dc.subjectT7 Replisome
dc.subjectDNA replication
dc.subjectDNA damage
dc.subjectFlow stretching single molecule assay
dc.subjectReplication fork
dc.titleEncounter of T7 Replisome with Abasic DNA Lesion
dc.typeThesis
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Division
dc.rights.embargodate2019-11-20
thesis.degree.grantorKing Abdullah University of Science and Technology
dc.contributor.committeememberMahfouz, Magdy M.
dc.contributor.committeememberFalqui, Andrea
thesis.degree.disciplineBioscience
thesis.degree.nameMaster of Science
dc.rights.accessrightsAt the time of archiving, the student author of this thesis opted to temporarily restrict access to it. The full text of this thesis became available to the public after the expiration of the embargo on 2019-11-20.
refterms.dateFOA2019-11-20T00:00:00Z
kaust.request.doiyes


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