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Protein dynamics during presynaptic-complex assembly on individual single-stranded DNA molecules

Nature Structural & Molecular Biology volume 21, pages 893–900 (2014)Cite this article

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Abstract

Homologous recombination is a conserved pathway for repairing double-stranded breaks, which are processed to yield single-stranded DNA overhangs that serve as platforms for presynaptic-complex assembly. Here we use single-molecule imaging to reveal the interplay between Saccharomyces cerevisiae RPA, Rad52 and Rad51 during presynaptic-complex assembly. We show that Rad52 binds RPA–ssDNA and suppresses RPA turnover, highlighting an unanticipated regulatory influence on protein dynamics. Rad51 binding extends the ssDNA, and Rad52–RPA clusters remain interspersed along the presynaptic complex. These clusters promote additional binding of RPA and Rad52. Our work illustrates the spatial and temporal progression of the association of RPA and Rad52 with the presynaptic complex and reveals a new RPA–Rad52–Rad51–ssDNA intermediate, with implications for how the activities of Rad52 and RPA are coordinated with Rad51 during the later stages of recombination.

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Figure 1: Single-stranded DNA curtain assay for presynaptic-complex assembly.
Figure 2: Individual Rad52 complexes binding to RPA–ssDNA.
Figure 3: Nucleation and growth of Rad52 on RPA–ssDNA.
Figure 4: Rad52 regulates RPA turnover.
Figure 5: Protein dynamics during presynaptic-complex assembly.
Figure 6: Assembly of Rad51–Rad52–RPA–ssDNA presynaptic intermediates.
Figure 7: Model for RPA and Rad52 dynamics during presynaptic-complex assembly.

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Acknowledgements

We thank L. Symington and members of the Greene and Sung laboratories for comments on the manuscript. This research was funded by the US National Institutes of Health grants GM074739 (E.C.G), RO1ES007061 (P.S.) and CA146940 (E.C.G. and P.S.). This work was partially supported by the Nanoscale Science and Engineering Initiative of the US National Science Foundation under award no. CHE-0641523 and by the New York State Office of Science, Technology, and Academic Research (NYSTAR). E.C.G. is supported as an Early Career Scientist by the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA

    Bryan Gibb & Eric C Greene

  2. Department of Biological Sciences, Columbia University, New York, New York, USA

    Ling F Ye

  3. Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut, USA

    YoungHo Kwon, Hengyao Niu & Patrick Sung

  4. Howard Hughes Medical Institute, Columbia University, New York, New York, USA

    Eric C Greene

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Contributions

B.G. and L.F.Y. cloned, expressed and purified RPA and Rad52, conducted the single-molecule experiments and analyzed the resulting data. Y.K. and H.N. expressed and purified Rad51, and Y.K. conducted bulk biochemical experiments. E.C.G. supervised the project. B.G. and E.C.G. wrote the manuscript with input from all coauthors.

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Correspondence to Eric C Greene.

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Integrated supplementary information

Supplementary Figure 1 Strand-annealing activity assays for fluorescent versions of Rad52.

(a) Schematic of strand annealing assay using complementary 83-nt ssDNA substrates, one of which was radiolabeled (red asterisk). Each substrate was mixed with RPA followed by Rad52, and the two reaction mixes were combined and incubated for the indicated times. (b) Products were resolved by 8% PAGE and (c) quantitated by phosphorimaging (error bars report the standard deviation of 3 experiments).

Supplementary Figure 2 Mediator activity assays for fluorescent versions of Rad52.

(a) Schematic for the mediator assay using a 150-nt ssDNA substrate that was mixed with RPA, Rad52, and Rad52, followed by the addition of a radiolabeled 40-bp dsDNA substrate. (b) Reactions were incubated for an additional 30 minutes, and reaction products were resolved by 8% PAGE and (c) quantitated by phosphorimaging (error bars report the standard deviation of 2-4 experiments). For quantitation, the amount of product formed in the presence of only Rad51 and RPA (lane 3), was subtracted from the amount of product formed in reactions with Rad52.

Supplementary Figure 3 Rad52 does not displace RPA from ssDNA.

A biotinylated 74-nt ssDNA oligo was bound to streptavidin magnetic beads and incubated with saturating amounts of wt RPA (400 nM). Increasing amounts of wt Rad52 were then added to the RPA-bound oligo in the presence of 400 nM free RPA and incubated for 20 minutes at 30 °C. The bound and free fractions were separated by pelleting the beads, and then resolved on an 8-16% SDS-PAGE gel and detected with Coomassie staining. Note that Rfa3 migrates off the gels under these electrophoresis conditions.

Supplementary Figure 4 Rad52 binding to sites occupied by preexisting Rad52 complexes.

(a) Positions of SNAP488-Rad52 (green) bound to a wtRPA-ssDNA complex. (b) Kymograph showing SNAP546-Rad52 (magenta) association with the SNAP488-Rad52/wtRPA/ssDNA complexes. (c) Line graphs showing integrated intensities of SNAP488-Rad52 and average integrated intensity over time for SNAP546-Rad52. The pixel positions for all three panels are co-aligned for direct comparison. (d-f) Same sets of data collected from a different ssDNA molecule.

Supplementary Figure 5 RPA-mCherry bound by wild-type Rad52 resists exchange when chased with wild-type RPA.

(a) Additional examples of kymographs showing RPA-mCherry turnover in the presence of wt RPA. (b) Examples of kymographs showing that the presence of wt Rad52 (0.6 − 1.0 nM) suppresses RPA-mCherry turnover when chased with wt RPA. (c) Examples of integrated RPA-mCherry signal over time (± 0.6 - 1.0 nM Rad52) after chasing with 100 nM wt RPA. The slower decrease in signal observed after the initial phase of rapid turnover is attributed to photo-bleaching.

Supplementary Figure 6 Co-occupancy of RPA, Rad52 and RPA on ssDNA.

Pull down assays performed with (a) ssDNA cellulose and (b) a 74-nt biotinylated oligo bound to streptavidin magnetic beads. The ssDNA was mixed buffer containing wt Rad51 (10 μM), wt Rad52 (1 μM), and wt RPA (0, 25, 50, 100, 200, and 400 nM) for 10 minutes at 37°C. Free and bound fractions were recovered and then resolved on an 8-16% SDS-PAGE gel and detected with Coomassie staining. A beads only negative control (labeled Beads or Beads NC) in (b) shows little protein binding to magnetic beads lacking ssDNA.

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Gibb, B., Ye, L., Kwon, Y. et al. Protein dynamics during presynaptic-complex assembly on individual single-stranded DNA molecules. Nat Struct Mol Biol 21, 893–900 (2014). https://doi.org/10.1038/nsmb.2886

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