Characterizing the Final Steps of Chromosomal Replication at the Single-molecule Level in the Model System Escherichia coli

Handle URI:
http://hdl.handle.net/10754/583819
Title:
Characterizing the Final Steps of Chromosomal Replication at the Single-molecule Level in the Model System Escherichia coli
Authors:
Elshenawy, Mohamed M. ( 0000-0002-8599-8388 )
Abstract:
In the circular Escherichia coli chromosome, two replisomes are assembled at the unique origin of replication and drive DNA synthesis in opposite directions until they meet in the terminus region across from the origin. Despite the difference in rates of the two replisomes, their arrival at the terminus is synchronized through a highly specialized system consisting of the terminator protein (Tus) bound to the termination sites (Ter). This synchronicity is mediated by the polarity of the Tus−Ter complex that stops replisomes from one direction (non-permissive face) but not the other (permissive face). Two oppositely oriented clusters of five Tus–Ters that each block one of the two replisomes create a “replication fork trap” for the first arriving replisome while waiting for the late arriving one. Despite extensive biochemical and structural studies, the molecular mechanism behind Tus−Ter polar arrest activity remained controversial. Moreover, none of the previous work provided answers for the long-standing discrepancy between the ability of Tus−Ter to permanently stop replisomes in vitro and its low efficiency in vivo. Here, I spearheaded a collaborative project that combined single-molecule DNA replication assays, X-ray crystallography and binding studies to provide a true molecular-level understanding of the underlying mechanism of Tus−Ter polar arrest activity. We showed that efficiency of Tus−Ter is determined by a head-to-head kinetic competition between rate of strand separation by the replisome and rate of rearrangement of Tus−Ter interactions during the melting of the first 6 base pairs of Ter. This rearrangement maintains Tus’s strong grip on the DNA and stops the advancing replisome from breaking into Tus−Ter central interactions, but only transiently. We further showed how this kinetic competition functions within the context of two mechanisms to impose permanent fork stoppage. The rate-dependent fork arrest activity of Tus−Ter explains its low efficiency in vivo and why contradictory in vitro results from previous studies have led to controversial elucidations of the mechanism. It also provides the first example where the intrinsic heterogeneity in rate of individual replisomes could have different biological outcomes in its communication with double-stranded DNA-binding protein barriers.
Advisors:
Hamdan, Samir
Committee Member:
Habuchi, Satoshi ( 0000-0002-6663-2807 ) ; Ooi, Boon S. ( 0000-0001-9606-5578 ) ; Magistretti, Pierre J. ( 0000-0002-6678-320X ) ; Dixon, Nicholas E.
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division
Program:
Bioscience
Issue Date:
Dec-2015
Type:
Dissertation
Appears in Collections:
Dissertations; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.advisorHamdan, Samiren
dc.contributor.authorElshenawy, Mohamed M.en
dc.date.accessioned2015-12-13T10:06:19Zen
dc.date.available2015-12-13T10:06:19Zen
dc.date.issued2015-12en
dc.identifier.urihttp://hdl.handle.net/10754/583819en
dc.description.abstractIn the circular Escherichia coli chromosome, two replisomes are assembled at the unique origin of replication and drive DNA synthesis in opposite directions until they meet in the terminus region across from the origin. Despite the difference in rates of the two replisomes, their arrival at the terminus is synchronized through a highly specialized system consisting of the terminator protein (Tus) bound to the termination sites (Ter). This synchronicity is mediated by the polarity of the Tus−Ter complex that stops replisomes from one direction (non-permissive face) but not the other (permissive face). Two oppositely oriented clusters of five Tus–Ters that each block one of the two replisomes create a “replication fork trap” for the first arriving replisome while waiting for the late arriving one. Despite extensive biochemical and structural studies, the molecular mechanism behind Tus−Ter polar arrest activity remained controversial. Moreover, none of the previous work provided answers for the long-standing discrepancy between the ability of Tus−Ter to permanently stop replisomes in vitro and its low efficiency in vivo. Here, I spearheaded a collaborative project that combined single-molecule DNA replication assays, X-ray crystallography and binding studies to provide a true molecular-level understanding of the underlying mechanism of Tus−Ter polar arrest activity. We showed that efficiency of Tus−Ter is determined by a head-to-head kinetic competition between rate of strand separation by the replisome and rate of rearrangement of Tus−Ter interactions during the melting of the first 6 base pairs of Ter. This rearrangement maintains Tus’s strong grip on the DNA and stops the advancing replisome from breaking into Tus−Ter central interactions, but only transiently. We further showed how this kinetic competition functions within the context of two mechanisms to impose permanent fork stoppage. The rate-dependent fork arrest activity of Tus−Ter explains its low efficiency in vivo and why contradictory in vitro results from previous studies have led to controversial elucidations of the mechanism. It also provides the first example where the intrinsic heterogeneity in rate of individual replisomes could have different biological outcomes in its communication with double-stranded DNA-binding protein barriers.en
dc.language.isoenen
dc.subjectSingle Molecule Imagingen
dc.subjectDNA Replicationen
dc.subjectReplication terminationen
dc.subjectChromosomal segregationen
dc.titleCharacterizing the Final Steps of Chromosomal Replication at the Single-molecule Level in the Model System Escherichia colien
dc.typeDissertationen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
thesis.degree.grantorKing Abdullah University of Science and Technologyen_GB
dc.contributor.committeememberHabuchi, Satoshien
dc.contributor.committeememberOoi, Boon S.en
dc.contributor.committeememberMagistretti, Pierre J.en
dc.contributor.committeememberDixon, Nicholas E.en
thesis.degree.disciplineBioscienceen
thesis.degree.nameDoctor of Philosophyen
dc.person.id101984en
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