Investigating RecA Filament Assembly in the Presence of SSB at Single-Molecule Level
Date Issued
2013
Date
2013
Author(s)
Tseng, Tzu-Ling
Abstract
RecA proteins are essential for DNA homologous recombination and DNA repair in E. Coli and other eubacteria. In the typical DNA strand-exchange reaction, the RecA assembles on single-stranded DNA. For function in the DNA strand-exchange reaction, RecA have to first bind to single-stranded DNA to form nucleoprotein filaments. Single-stranded DNA generated in the cell during DNA metabolism is stablized and protected by binding of single-stranded DNA binding proteins. The formation of RecA filaments requires SSB protein to be displaced from the ssDNA. Project to monitor the SSB functioning as a barrier to RecA nucleation on ssDNA or RecA actively involves in the displacement of SSB from the ssDNA.
The mechanism of efficient SSB displacement by RecA was unclear given the tight binding of SSB to ssDNA. In order to comparing EcRecA assembly on ssDNA in the presence or absence of SSB have different function, we introduced a specific mutant RecA E38K encodes a form of RecA protein which is able to achieve the nucleation reaction without the help of RecA loading mediators RecFOR and exhibits constitutive SOS expression. We used single molecular tethered particle motion (TPM) techniques to directly observe the assembly dynamics of RecA proteins on individual DNA molecules by observing changes in Brownian motion (BM) produced by RecA binding. Without secondary structure gapped ssDNA substrates could exclude random sequence DNA forming secondary structure on BM detection inaccuracy. Diffusional migration of SSB on ssDNA provides the region for RecA nucleate on SSB-free ssDNA, then RecA proteins monomer-monomer extension on ssDNA to form RecA nucleoprotein filament. We demonstrated that EcRecA needs six subunits to nucleate on ssDNA, but E38K mutant only needs less than six subunits to nucleate. The extension function and nucleoprotein filament formation are similar both on EcRecA and E38K mutant.
Subjects
RecA
RecA突變體
單股DNA結合蛋白
同源重組修復
單分子拴球實驗
Type
thesis
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