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

Fagerburg, Matthew (2011) Single-Molecule Insights into PcrA-Driven Disruption of RecA Filaments. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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
Status: Unpublished
CreatorsEmailPitt UsernameORCID
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorLeuba, SanfordLEUBA@pitt.eduLEUBA
Committee MemberOpresko, Patriciaplo4@pitt.eduPLO4
Committee MemberVan Houten, Bennettvanhoutenb@upmc.eduBEV15
Committee MemberSteinman, Richardsteinman@pitt.eduSTEINMAN
Date: 19 December 2011
Date Type: Publication
Defense Date: 9 December 2011
Approval Date: 19 December 2011
Submission Date: 15 December 2011
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 154
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Biophysics and Structural Biology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: single molecule, FRET, PcrA, RecA, recombination
Date Deposited: 19 Dec 2011 19:50
Last Modified: 19 Dec 2016 14:38


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