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
Type
ThesisAuthors
Shirbini, Afnan
Advisors
Hamdan, Samir
Committee members
Arold, Stefan T.
Saikaly, Pascal

Program
BioscienceDate
2017-05Embargo End Date
2017-05-09Permanent link to this record
http://hdl.handle.net/10754/623462
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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-V6QACae974a485f413a2113503eed53cd6c53
10.25781/KAUST-V6QAC