Single-molecule FRET study on the RecA-mediated DNA repair
Joo, Chirlmin
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https://hdl.handle.net/2142/34767
Description
Title
Single-molecule FRET study on the RecA-mediated DNA repair
Author(s)
Joo, Chirlmin
Issue Date
2007-05
Doctoral Committee Chair(s)
Ha, Taekjip
Department of Study
Physics
Discipline
Physics
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Single-Molecule FRET
RecA Filament
RecA-Mediated Strand Exchange
Language
en
Abstract
The RecA protein helps maintain genomic integrity through recombination. Using single-molecule assays and hidden Markov modeling, we show the most direct evidence that a
RecA filament grows and shrinks primarily, one monomer at a time only from the extremities. Both ends grow and shrink, contrary to earlier predictions, and a higher binding rate at one end is responsible for directional filament growth. Quantitative rate determination also provides insights into how RecA might control DNA accessibility in vivo.
Next, we estimate that about five monomers are sufficient for filament nucleation.
Using a vesicle encapsulation assay, we find that the nucleation is likely to occur through the binding of a pre-assembled oligomer rather than the simultaneous binding of several monomers. Although normally SSB prevents filament nucleation, single RecA monomers can easily be added to a pre-existing filament and displace SSB from DNA at the rate of filament extension. This supports the proposal for a passive role of RecA-loading machineries in SSB removal.
The RecA filament mediates homologous strand exchange resulting in the four-way DNA junction in vivo. We develop single-molecule assays to observe the strand exchange in real time to understand its molecular mechanism.
Finally, we explore the conformational repertoire of the four-way junction by characterizing the effects of metal ions. And we find that the four arms’ movements are synchronized to each other during conformational dynamics. We also seek to detect possible rare species such as intermediate and parallel conformations. While our study suggests that the intermediate form is achieved very frequently, we cannot detect even a transient existence of a parallel form.
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