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

Handle URI:
http://hdl.handle.net/10754/623462
Title:
Towards observing the encounter of the T7 DNA replication fork with a lesion site at the Single molecule level
Authors:
Shirbini, Afnan ( 0000-0002-3559-7594 )
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.
Advisors:
Hamdan, Samir M ( 0000-0001-5192-1852 )
Committee Member:
Arold, Stefan T. ( 0000-0001-5278-0668 ) ; Saikaly, Pascal ( 0000-0001-7678-3986 )
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division
Program:
Bioscience
Issue Date:
May-2017
Type:
Thesis
Appears in Collections:
Theses

Full metadata record

DC FieldValue Language
dc.contributor.advisorHamdan, Samir Men
dc.contributor.authorShirbini, Afnanen
dc.date.accessioned2017-05-10T08:19:36Z-
dc.date.available2017-05-10T08:19:36Z-
dc.date.issued2017-05-
dc.identifier.urihttp://hdl.handle.net/10754/623462-
dc.description.abstractSingle-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.en
dc.language.isoenen
dc.subjectDNA Damageen
dc.subjectabasicen
dc.subjectT7 replicationen
dc.subjectbypassen
dc.subjectSingle-moleculeen
dc.subjectgp5 - gp4en
dc.titleTowards observing the encounter of the T7 DNA replication fork with a lesion site at the Single molecule levelen
dc.typeThesisen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
thesis.degree.grantorKing Abdullah University of Science and Technologyen_GB
dc.contributor.committeememberArold, Stefan T.en
dc.contributor.committeememberSaikaly, Pascalen
thesis.degree.disciplineBioscienceen
thesis.degree.nameMaster of Scienceen
dc.person.id144593en
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