Figure 6. Protein-protein interactions

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Figure 6. Protein-protein interactions Figure 6. Protein-protein interactions. (A) Detection of direct protein-protein interactions by His6-affinity ... Figure 6. Protein-protein interactions. (A) Detection of direct protein-protein interactions by His6-affinity pulldowns: purified Dmc1 (D), Mei5-Sae3 (M), Rad51-II3A (R), Hop2-Mnd1 (H), and Rdh54/Tid1 (T) were His6-tagged. Each of these proteins was first incubated individually with untagged RPA, followed by the addition of Ni sepharose beads to capture the interacting protein complex through the His6-tag. The Ni captured complexes were eluted with a solution containing 1.7% SDS and 0.1 M DTT and separated on 12% SDS-PAGE, further analyzed by Western blotting using antibodies against all the proteins involved in the analysis for detection. An asterisk indicates the position of RPA2 (A2) pulled down by Dmc1 and Mei5-Sae3. (B) Detection of direct protein-protein interactions by in vitro co-immunoprecipitation of anti-RPA2 antibody with purified proteins as indicated. (C) Detection of direct protein-protein interactions by in vitro co-immunoprecipitation with anti-Dmc1 antibody. A mixture of antibodies against all proteins was used for protein detection. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 2, 20 November 2018, Pages 747–761, https://doi.org/10.1093/nar/gky1160 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 1. In vitro reconstitution of Dmc1-dependent recombination with five accessory proteins. (A) Scheme for the ... Figure 1. In vitro reconstitution of Dmc1-dependent recombination with five accessory proteins. (A) Scheme for the 2-step D-loop assay with the pre-mixed reaction regimen. For the first step, reaction components, including pre-mixed proteins, were incubated with ssDNA at 37°C for 5 min to make Dmc1 filaments. The second step is initiated by addition of target dsDNA followed by incubation for 30 min to form D-loops. (B) D-loop assay reactions using the 90C ssDNA (30 nM or 2.7 μM nt) and various mixtures of proteins. Abbreviations for proteins and their concentrations in the reactions: D = Dmc1 (3 μM), A = RPA (0.2 μM), M = Mei5-Sae3 (0.5 μM), R = Rad51-II3A (0.25 μM), H = Hop2-Mnd1 (0.2 μM), T = Rdh54/Tid1 (0.1 μM); dsDNA was supercoiled plasmid pRS306 (5 nM or 22 μM bp). (C) D-loop assay reactions using the 563 nt ssDNA (4 nM or 2.2 μM nt) and various combinations of accessory proteins at the same concentrations as in B, dsDNA was at 2.5 nM (11 μM bp). ‘L’ stands for position of the largest D-loops detected, and ‘s’ for position of the smallest D-loops. The D-loop yield was expressed as a percentage of input plasmid DNA. The gel images from D-loop assays were shown and data were quantified and graphed (n = 3, ± s.e.m.). [NB: Pre-mixing results in lower levels of D-loop activity than observed when proteins are added sequentially to ssDNA, accounting for the difference in D-loop yields seen here as compared to our previously published papers (11,16)]. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 2, 20 November 2018, Pages 747–761, https://doi.org/10.1093/nar/gky1160 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2. Effects of RPA on secondary structure and inhibitory binding of non-homologous ssDNA to Dmc1-Site II. (A) ... Figure 2. Effects of RPA on secondary structure and inhibitory binding of non-homologous ssDNA to Dmc1-Site II. (A) Cartoons of predicted secondary structures of 90C ssDNA (significant secondary structure) and 90NF ssDNA (no secondary structure at 37°C). [NB: the cartoon for 90NF was produced by a computer program using 25°C rather then 37°C as the input temperature because the program does not produce an output image if no secondary structure is detected. The small stem loop shown in the cartoon is not predicted to form at 37°C.]. (B) D-loop formation by Dmc1 was examined using 90C ssDNA (30 nM or 2.7 μM nt) and 90NF ssDNA (30 nM or 2.7 μM nt). The pre-mixed regimen was used. Abbreviations and concentrations were as in Figure 1. (C) Competition of RPA with Dmc1-Site II for ssDNA binding. In the pre-mixed regimen, all six proteins (ADRMHT) were first added to 90C ssDNA (1× = 30 nM or 2.7 μM nt) to make Dmc1-ssDNA filaments. After filament formation, increasing amounts of non-homologous ssDNA (90NF, 1x = 30 nM or 2.7 μM nt) were added to reactions 5–10 as indicated to allow binding to the secondary Site II in Dmc1-ssDNA filaments. RPA (1× = 0.2 μM) was then added in increasing amounts to reactions 8–10 before the addition of plasmid pRS306 to each sample to initiate D-loop formation (n = 3, ± s.e.m.). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 2, 20 November 2018, Pages 747–761, https://doi.org/10.1093/nar/gky1160 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. Dmc1 reactions lacking RPA are more efficient when Hop2-Mnd1 and Rdh54/Tid1 are added late to reactions. (A) ... Figure 3. Dmc1 reactions lacking RPA are more efficient when Hop2-Mnd1 and Rdh54/Tid1 are added late to reactions. (A) The order of addition of reaction components is as indicated. The concentrations of reactants are as detailed in Figure 1 and in Materials and Methods. The first arrow indicates incubation was at 37°C for 10 min followed by the second arrow, which indicates an additional incubation at 37°C for 15 min. Hop2-Mnd1 and dsDNA in reaction 1 were incubated at room temperature for 3 min prior to adding to Dmc1-ssDNA filaments. The gel images from D-loop assays are shown and results graphed (n = 3, ± s.e.m.). Data shown here was published previously (16) and are presented for comparison to B with permission [NB: permission pending]. (B) The same experimental procedure as in A except Rdh54/Tid1 was used in place of Hop2-Mnd1. (C) Deletion and add-back reactions in the pre-mixed regimen of Hop2-Mnd1 or Rdh54/Tid1 in the absence of RPA. The order of addition of components were as indicated. Abbreviations for proteins and protein and DNA concentration are as in Figure 1. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 2, 20 November 2018, Pages 747–761, https://doi.org/10.1093/nar/gky1160 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. Effect of RPA in pre-staged reactions Figure 4. Effect of RPA in pre-staged reactions. D-loop formation by Dmc1 and its accessory proteins was examined using ... Figure 4. Effect of RPA in pre-staged reactions. D-loop formation by Dmc1 and its accessory proteins was examined using 90C ssDNA and 90NF ssDNA (30 nM or 2.7 μM nt). Reactions were with or without RPA as indicated. Abbreviations and reaction component concentrations were as in Figure 1 (n = 3, ±s.e.m.). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 2, 20 November 2018, Pages 747–761, https://doi.org/10.1093/nar/gky1160 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5. RPA outcompetes Hop2-Mnd1 for binding to ssDNA Figure 5. RPA outcompetes Hop2-Mnd1 for binding to ssDNA. (A) Scheme for bead ‘catch and release’ of protein complexes ... Figure 5. RPA outcompetes Hop2-Mnd1 for binding to ssDNA. (A) Scheme for bead ‘catch and release’ of protein complexes bound to ssDNA. db-ssDNA stands for desthiobiotin modified ssDNA. (B) The captured protein complexes on ssDNA were analyzed by Western blotting. RPA were 0.125, 0.25 and 0.5 μM in lanes 1–3; 0.5 μM in lanes 7–13. Hop2-Mnd1 were 0.2, 0.4 and 0.6 μM in lanes 4–6, lanes 7–9, and lanes 10–12. Lane 13 is a control that lacked db-ssDNA but contained 0.5 μM RPA and 0.4 μM Hop2-Mnd1. (C) The unbound protein complexes analyzed by western blotting. (D) The eluted db-ssDNA–protein complexes were denatured and analyzed for DNA content on 8% urea-PAGE via staining with SYBR-gold. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 2, 20 November 2018, Pages 747–761, https://doi.org/10.1093/nar/gky1160 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 7. Model. (1) RPA binds ssDNA thereby removing secondary structure and preventing binding of Hop2-Mnd1 and ... Figure 7. Model. (1) RPA binds ssDNA thereby removing secondary structure and preventing binding of Hop2-Mnd1 and Rdh54/Tid1. (2) RPA carries out hand-off reactions allowing Rad51+Mei5-Sae3 to assist Dmc1 filament formation. The protein configuration shown is speculative but depicts potentially relevant protein-protein interactions between RPA and Mei5-Sae3, RPA and Dmc1, Rad51 and Mei5-Sae3, Dmc1 and Mei5-Sae3, and, particularly importantly, between Dmc1 and Rad51. (3) dsDNA bound Hop2-Mnd1 captures filaments via homology-independent interactions with Dmc1. (4) Homology recognition occurs to form a homology-dependent nascent D-loop which is stabilized by RPA binding to the displaced ssDNA strand. (5) Rdh54/Tid1 binds the D-loop, via contacts with both ssDNA and dsDNA and then translocates across the D-loop stabilizing it by displacing Dmc1 and elongating the D-loop. RPA can elongate the D-loop, even in the absence of Rdh54/Tid1, but does not afford the stability associated with Dmc1 displacement and/or simultaneous binding by Rdh54/Tid1 to both ssDNA and dsDNA. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 47, Issue 2, 20 November 2018, Pages 747–761, https://doi.org/10.1093/nar/gky1160 The content of this slide may be subject to copyright: please see the slide notes for details.