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    Encounter of T7 Replisome with Abasic DNA Lesion

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    Name:
    Lubna Alhudhali Thesis.pdf
    Size:
    3.550Mb
    Format:
    PDF
    Description:
    MS Thesis
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    Type
    Thesis
    Authors
    Alhudhali, Lubna F. cc
    Advisors
    Hamdan, Samir cc
    Committee members
    Mahfouz, Magdy M. cc
    Falqui, Andrea cc
    Program
    Bioscience
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Date
    2019-11
    Embargo End Date
    2019-11-20
    Permanent link to this record
    http://hdl.handle.net/10754/660187
    
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    Access Restrictions
    At 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.
    Abstract
    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.
    Citation
    Alhudhali, L. F. (2019). Encounter of T7 Replisome with Abasic DNA Lesion. KAUST Research Repository. https://doi.org/10.25781/KAUST-01928
    DOI
    10.25781/KAUST-01928
    ae974a485f413a2113503eed53cd6c53
    10.25781/KAUST-01928
    Scopus Count
    Collections
    Biological and Environmental Sciences and Engineering (BESE) Division; Bioscience Program; Theses

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