Direct Observation of RecA-Mediated Four-strand Exchange at the Single-Molecule Level
Date Issued
2010
Date
2010
Author(s)
Hsu, Mu-Ni
Abstract
E. coli RecA protein is required for repairing damaged DNA through homologous recombination pathway. The assembled RecA/DNA nucleoprotein filaments catalyze the reactions that paired and exchange with homologous DNA sequence. We used single-molecule tethered particle motion (TPM) experiments to directly observe the strand exchange process catalyzed by RecA in real-time. The surface-bound DNA is a duplex DNA with a bead could be displaced due to four-strand exchange. Calculate the percentage of leaving tethers under different condition to obtain the properties of four-strand exchange. The RecA-mediated strand exchange efficiency is higher for ATP than ATPγS, suggesting that ATP hydrolysis by RecA is required to complete strand
exchange. In addition, the RecA-mediate strand exchange is shown to progress either from 5’end or from 3’end in our experiment. Furthermore quantitative analysis of DNA exchanging time courses using ‘‘n-step’’ sequential mechanisms, can reveal information about the rate constant of four-strand exchange reaction and the average number of basepairs in the strand exchange cycle. The result indicates that the rate is larger for longer single-stranded gap and 5’overhang.
Subjects
RecA
four-strand exchange
DNA repair
homologous recombination
TPM
Type
thesis
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