Research ArticleSTRUCTURAL BIOLOGY

RecA filament maintains structural integrity using ATP-driven internal dynamics

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Science Advances  06 Sep 2017:
Vol. 3, no. 9, e1700676
DOI: 10.1126/sciadv.1700676
  • Fig. 1 Real-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).

  • Fig. 2 Chi sequence–dependent phase bias.

    (A) DNA designs with different distances between the Chi sequence and the dye pair. Interdye separation was kept at 10 nt, whereas dye-to-Chi distances were increased by 1 nt from 9 to 11 nt. (B) Population distributions obtained from the 9-, 10-, and 11-nt dye-to-Chi distances. The measured population ratios (low to high) were 1:5, 1.6:1, and 1:5 for 9-, 10-, and 11-nt dye-to-Chi distances, respectively. Note that these three DNA samples are exactly the same as the one used in Fig. 1E (10 nt) except for the varying distance of the Chi sequences. (C) DNA designs for longer dye-to-Chi distances of 10, 85, and 160 nt. (D) The population distributions obtained from the 10-, 85-, and 160-nt dye-to-Chi distances. The measured population ratios (low to high) were 2.0:1, 1.4:1, and 1.1:1 for 10-, 85-, and 160-nt dye-to-Chi distances, respectively. (E) The population ratios of the low FRET peak to the high FRET peak (black rectangle). Error bars are SEs from three independent data sets. Gray and orange solid lines are linear and single-exponential fits to the data. Dashed line represents the 1:2 population ratio limit of the random phase distribution. All the measurements in (B) to (E) were carried out in the presence of 1 mM ATP.

  • Fig. 3 Phase bias due to 3-nt-period sequence pattern.

    (A) DNA designs with different distances (5, 6, and 7 nt) between TGG repeats and a dye pair. The interdye separation was kept at 10 nt. (B) The population distributions obtained from the 5-, 6-, and 7-nt dye-to-TGG separations. The population ratios (low to high FRET state) determined by Gaussian fitting of each peak are 1:3.3, 1.5:1, and 1:3.3 for 5-, 6-, and 7-nt dye-to-Chi distances, respectively. (C) Schematic of the base grouping and gap generation in which the bases are grouped into “TGG” that account for the data in (B). Each DNA shows a different number of the gaps between the two dyes depending on their dye-to-TGG distance. (D) DNA designs with different base distributions. In the sequence named “islands,” the four triplets of TGG were distributed along the DNA, whereas their positions from the dye pairs are kept in phase. In the sequence “dispersed,” the guanine bases were displaced further, whereas their positions are kept in phase. (E) The population distributions obtained from the islands and dispersed. The population ratios (low to high FRET state) determined by Gaussian fitting of each peak are 1.8 and 1.6 with islands and dispersed, respectively. (F) DNA designs with different triplet sequences placed 6 nt apart from the dye pair of a 10-nt separation. (G) The population ratio (low FRET to high FRET) observed from the four triplet sequences in (F). Dashed line indicates the 1:2 ratio limit expected from the random distribution. (H) Correlation plot between the population ratios in (G) and the ATP hydrolysis rates measured from the filament formed on the triplet repeats. The ATP hydrolysis rates were adapted from the study of Kim et al. (38). All the measurements were carried out in the presence of 1 mM ATP.

  • Fig. 4 Suppression of phase shift dynamics from 5′ side of a RecA filament.

    (A) Schematic of the heterologous RecA filament construct. A portion of the RecA filament was formed with ATPγS [formed on double-stranded DNA (dsDNA) region] and was connected to the filament in the 3′ direction formed with ATP (on ssDNA region). The interdye separation was 10 nt. (B) Single-molecule FRET distribution obtained from the heterologous RecA filaments. Population ratio (low to high) was 1:2.3. (C and D) Representative single-molecule time traces of (C) up-stable and (D) down-stable filaments. Time traces with 32-ms time resolution (gray lines) were depicted with their moving average with a 1-s time window (black lines). (E) Dwell time distribution of up state (left) and down state (right) from the transitions found in up-stable traces. The average dwell times were obtained by single-exponential fit to the data (red lines) from three independent data sets. (F) Dwell time distribution of down state (left) and up state (right) from the transitions found in down-stable traces. The average dwell times were obtained by single-exponential fit to the data (red lines) from three independent data sets.

  • Fig. 5 A model for the RecA filament formation working in concert with a RecA loading machinery.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/9/e1700676/DC1

    fig. S1. Single-molecule RecA filament phase detection assay.

    fig. S2. Single-molecule FRET time traces in the presence of ATP.

    fig. S3. Single-molecule FRET time traces in the presence of ATPγS.

    fig. S4. Dwell time distribution determined by hidden Markov method.

    fig. S5. Possible FRET distributions of the three DNA construct at each phase.

    fig. S6. The population distributions obtained from different triplet sequences in Fig. 3F.

    fig. S7. RecA ATPγS/ATP heterologous filament assay.

    table S1. Fit parameters obtained from the correlation functions in Fig. 1K.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Single-molecule RecA filament phase detection assay.
    • fig. S2. Single-molecule FRET time traces in the presence of ATP.
    • fig. S3. Single-molecule FRET time traces in the presence of ATPγS.
    • fig. S4. Dwell time distribution determined by hidden Markov method.
    • fig. S5. Possible FRET distributions of the three DNA construct at each phase.
    • fig. S6. The population distributions obtained from different triplet sequences in Fig. 3F.
    • fig. S7. RecA ATPγS/ATP heterologous filament assay.
    • table S1. Fit parameters obtained from the correlation functions in Fig. 1K.

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