LukeViews: 162, 2017.09.11 08:34:54
- Science Advances Vol. 3, no. 9, e1700676 (2017)
Sung Hyun Kim, TakKyoon Ahn, Tao Ju Cui, Sweeny Chauhan, Jaeyoung Sung, Chirlmin Joo, and Doseok Kim*
†Department of Physics and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, Republic of Korea.
‡Department of Bionanoscience, Kavli Institute of NanoScience, Delft University of Technology, 2628 CJ Delft, Netherlands.
§Department of Chemistry, Chung-Ang University, Seoul, Republic of Korea.
At the core of homologous DNA repair, RecA catalyzes the strand exchange reaction. This process is initiated by a RecA loading protein, which nucleates clusters of RecA proteins on single-stranded DNA. Each cluster grows to cover the single-stranded DNA but may leave 1- to 2-nucleotide (nt) gaps between the clusters due to three different structural phases of the nucleoprotein filaments. It remains to be revealed how RecA proteins eliminate the gaps to make a seamless kilobase-long filament. We develop a single-molecule fluorescence assay to observe the novel internal dynamics of the RecA filament. We directly observe the structural phases of individual RecA filaments and find that RecA proteins move their positions along the substrate DNA to change the phase of the filament. This reorganization process, which is a prerequisite step for interjoining of two adjacent clusters, requires adenosine triphosphate hydrolysis and is tightly regulated by the recombination hotspot, Chi. Furthermore, RecA proteins recognize and self-align to a 3-nt-period sequence pattern of TGG. This sequence-dependent phase bias may help the RecA filament to maintain structural integrity within the kilobase-long filament for accurate homology search and strand exchange reaction.
Fig. 1Real-time observation of RecA filament phase shifting dynamics.
(A) Schematic of phase detection of RecA filament. The number of the gaps between a dye pair is determined by the phase of the filament and can be read by FRET efficiency. (B) Schematic of DNA immobilization on the polyethylene glycol–coated quartz slide via a streptavidin-biotin linker. (C to E) Population distributions of single-molecule FRET from (C) an 8-nt, (D) a 9-nt, and (B) a 10-nt separation of the dye pair. Gray bars are the distribution observed in the presence of 1 μM RecA and 1 mM ATP, and empty bars are obtained from bare DNA in the absence of RecA. Solid lines are Gaussian fits. (F to H) Representative single-molecule traces obtained from (F) an 8-nt, (G) a 9-nt, and (H) a 10-nt dye separation. Time traces with 32-ms time resolution (gray) were depicted with their moving average with a 1-s time window (black). (I to J) Dwell time distributions of high (I) and low (J) FRET states with a 10-nt dye separation. Solid lines are single-exponential fits. A manually given threshold value was applied to each trace to determine the transition points, and 150 traces were processed to build a dwell time histogram. The average dwell times with SEs were obtained from three independent data sets. (K) Averaged time correlation functions of single-molecule traces obtained with 8 nt (orange filled circle), 9 nt (black open circle), and 10 nt (blue filled box) in the presence of ATP and 10 nt in the presence of ATPγS (gray crosses). Solid lines are two exponential (gray) and three exponential fits (red) of the correlation functions (fit parameters are summarized in table S1).