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Single-Molecule Insights into PcrA-Driven Disruption of RecA Filaments

Fagerburg, Matthew Single-Molecule Insights into PcrA-Driven Disruption of RecA Filaments. Doctoral Dissertation, University of Pittsburgh.

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    Abstract

    Homologous recombination (HR) plays a critical role in many important cellular processes, including the resolution of stalled replication forks. HR must be highly regulated within the cell because aberrant recombination can introduce gene deletions as well as structural barriers to genetic replication and repair; various families of proteins have evolved in different organisms to achieve this regulation. The bacterial DNA-binding protein RecA is one such prototypical agent that promotes HR. It forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) that act as HR loci. The assembly and disassembly of RecA filaments are dynamic, and depend on the ATPase cycle of the protein. Both processes are subject to modulation and regulation by other factors. RecA filaments can be actively removed from DNA by non-replicative helicases such as PcrA (present in Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumoniae) and UvrD (present in Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa), and deletion of either of these leads to dysregulation of HR, which suggests that they play an important role in regulating HR via the removal of RecA filaments. We used single-molecule FRET (smFRET) to further investigate this removal and discovered that the ATPase activity of RecA is required for it to occur. The exquisite sensitivity of the single molecule technique allowed us to observe individual, short RecA filaments on ssDNA, as well as how PcrA disrupts them. The work described in this dissertation highlights a novel mechanistic component in the regulation of RecA, namely the crucial role that its ATPase activity plays in filament removal by PcrA.


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    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmail
    Thesis AdvisorLeuba, SanfordLEUBA@pitt.edu
    Committee MemberOpresko, Patriciaplo4@pitt.edu
    Committee MemberVan Houten, Bennettvanhoutenb@upmc.edu
    Committee MemberSteinman, Richardsteinman@pitt.edu
    Title: Single-Molecule Insights into PcrA-Driven Disruption of RecA Filaments
    Status: Published
    Abstract: Homologous recombination (HR) plays a critical role in many important cellular processes, including the resolution of stalled replication forks. HR must be highly regulated within the cell because aberrant recombination can introduce gene deletions as well as structural barriers to genetic replication and repair; various families of proteins have evolved in different organisms to achieve this regulation. The bacterial DNA-binding protein RecA is one such prototypical agent that promotes HR. It forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) that act as HR loci. The assembly and disassembly of RecA filaments are dynamic, and depend on the ATPase cycle of the protein. Both processes are subject to modulation and regulation by other factors. RecA filaments can be actively removed from DNA by non-replicative helicases such as PcrA (present in Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumoniae) and UvrD (present in Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa), and deletion of either of these leads to dysregulation of HR, which suggests that they play an important role in regulating HR via the removal of RecA filaments. We used single-molecule FRET (smFRET) to further investigate this removal and discovered that the ATPase activity of RecA is required for it to occur. The exquisite sensitivity of the single molecule technique allowed us to observe individual, short RecA filaments on ssDNA, as well as how PcrA disrupts them. The work described in this dissertation highlights a novel mechanistic component in the regulation of RecA, namely the crucial role that its ATPase activity plays in filament removal by PcrA.
    Defense Date: 09 December 2011
    Approval Date: 19 December 2011
    Submission Date: 15 December 2011
    Release Date: 19 December 2011
    Access Restriction: No restriction; Release the ETD for access worldwide immediately.
    Patent pending: No
    Number of Pages: 154
    Institution: University of Pittsburgh
    Thesis Type: Doctoral Dissertation
    Refereed: Yes
    Degree: PhD - Doctor of Philosophy
    Uncontrolled Keywords: single molecule, FRET, PcrA, RecA, recombination
    Schools and Programs: School of Medicine > Molecular Biophysics and Structural Biology
    Date Deposited: 19 Dec 2011 14:50
    Last Modified: 20 Dec 2011 01:15

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