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    Towards observing the encounter of the T7 DNA replication fork with a lesion site at the Single molecule level

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    MS Thesis_Afnan Shirbini_Spring 2017.pdf
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    MS Thesis_Afnan Shirbini_Spring 2017
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    Type
    Thesis
    Authors
    Shirbini, Afnan cc
    Advisors
    Hamdan, Samir cc
    Committee members
    Arold, Stefan T. cc
    Saikaly, Pascal cc
    Program
    Bioscience
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Date
    2017-05
    Embargo End Date
    2017-05-09
    Permanent link to this record
    http://hdl.handle.net/10754/623462
    
    Metadata
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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 2017-05-09.
    Abstract
    Single-molecule DNA flow-stretching assays have been a powerful approach to study various aspects on the mechanism of DNA replication for more than a decade. This technique depends on flow-induced force on a bead attached to a surface-tethered DNA. The difference in the elastic property between double-strand DNA (long) and single-strand DNA (short) at low regime force allows the observation of the beads motion when the dsDNA is converted to ssDNA by the replisome machinery during DNA replication. Here, I aim to develop an assay to track in real-time the encounter of the bacteriophage T7 replisome with abasic lesion site inserted on the leading strand template. I optimized methods to construct the DNA substrate that contains the abasic site and established the T7 leading strand synthesis at the single molecule level. I also optimized various control experiments to remove any interference from the nonspecific interactions of the DNA with the surface. My work established the foundation to image the encounter of the T7 replisome with abasic site and to characterize how the interactions between the helicase and the polymerase could influence the polymerase proofreading ability and its direct bypass of this highly common DNA damage type.
    Citation
    Shirbini, A. (2017). Towards observing the encounter of the T7 DNA replication fork with a lesion site at the Single molecule level. KAUST Research Repository. https://doi.org/10.25781/KAUST-V6QAC
    DOI
    10.25781/KAUST-V6QAC
    ae974a485f413a2113503eed53cd6c53
    10.25781/KAUST-V6QAC
    Scopus Count
    Collections
    Biological and Environmental Sciences and Engineering (BESE) Division; Bioscience Program; Theses; Theses

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