1. Volume 29, Issue 2, Pages 243-254 (February 2008)
The Srs2 Helicase Activity Is Stimulated by Rad51 Filaments on dsDNA: Implications for Crossover Incidence during Mitotic Recombination 
Pauline Dupaigne, Cyrille Le Breton, Francis Fabre, Serge Gangloff, Eric Le Cam, Xavier Veaute 
Molecular Cell 
Volume 29, Issue 2, Pages (February 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
Models of Double-Stranded Break Repair by Homologous Recombination
Recombination is initiated by nucleolytic processing of the DSB to generate 3′ single-stranded tails that are rapidly covered with RPA. Rad51 recruitment displaces RPA leading to the formation of the presynaptic filament, which searches for an intact homologous template and then catalyzes invasion of the ssDNA into the donor molecule to form a D loop. The invading strand serves as a primer for DNA synthesis to extend the heteroduplex. Different pathways may further process this intermediate. The elongated invading strand can be displaced from the D loop and then anneals to the second end of the DSB (SDSA pathway). A new step of DNA synthesis and ligation leads to repair without production of CO. Alternatively, the capture of the second end results in the formation of a dHJ. dHJ intermediates can be subsequently resolved either by the dissolution pathway, which creates noncrossover products, or by endonucleolytic cleavage of the two Holliday junctions to generate CO or noncrossover products.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Srs2 Preferentially Unwinds PX Junction
(A) Scheme of a D loop intermediate. DNA in the gray box represents the structure called the PX junction.
(B) Helicase assay was performed with 62.5 nM Srs2 at 25°C.
(C) Quantification of the reactions shown in (B).
(D) Srs2 binds forked DNA better than PX junction as observed by gel shift assay.
(E) Quantification of the reactions shown in (D). The average values from four independent experiments in (C) and two in (E) were plotted. Error bars show the standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Srs2 Unwinds a dsDNA Covered with Rad51
(A) Experimental scheme.
(B) Increasing amounts of Srs2 were added after formation of a Rad51 nucleoprotein filament. Numbers at the bottom of the gel represent the percentage of unwinding.
(C) Reaction products examined by EM. (Ca) Rad51 nucleoprotein filament before incubation with Srs2. (Cb) Preformed Rad51 nucleoprotein filaments with 3′-tailed dsDNA were incubated with 125 nM Srs2. (Cc) Blow up of a molecule observed in (Cb). (Cd) Drawing of the molecule observed in (Cc). (Ce) Preformed Rad51 nucleoprotein filaments with 5′-tailed dsDNA were incubated with 125 nM Srs2. (Cf) Blow up of a molecule observed in (Ce). (Cg) Drawing of the molecule observed in (Cc). (Ch) Preformed Rad51 nucleoprotein filaments with 3′-tailed dsDNA were incubated with 1 μM Srs2. (Ci) Blow up of a partially unwound intermediate observed after incubation with 1 μM Srs2. (Cj) Drawing of the molecule observed in (Ci). Scale bars, 100 nm.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Stimulation of the Srs2-Unwinding Activity by Rad51
(A) In this assay, a 200 bp duplex with a 3′ ssDNA tail of 409 nt was used. The drawings above each lane indicate the state of the majority of the molecules just before the addition of Srs2 to the reaction.
(B) Quantification of the helicase assay. Results were expressed as the percentage of total substrate unwound. The data presented are the average of three independent experiments. Error bars show the standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Srs2 Translocates on RPA-Covered ssDNA
(A) Scheme on the top indicates the order of introduction of the different proteins to obtain 3′-tailed dsDNA substrates with Rad51 on the duplex portion and RPA on the ssDNA. Srs2 is able to unwind dsDNA in these complexes. The average values from two independent experiments were plotted. Error bars show the standard deviation.
(B) Srs2 via its translocase activity disrupts the biotin-streptavidin interaction.
(C) EM analyses reveal that Srs2 translocates on RPA-covered ssDNA. (Ca) RPA bound to streptavidin-bound 5′-biotinylated 609 oligonucleotides. (Cb) 5′-biotinylated 609 bp blunt-ended DNA. (Cc) The dsDNA was used as a nanoscale biopointer (gray triangle) bound to the RPA-ssDNA (white triangle) via a complex. (Cd) Blow up of a molecule observed after formation of the complex. (Ce) Srs2 after translocation on RPA-ssDNA disrupts the biotin-streptavidin interaction and separates the dsDNA from the RPA-ssDNA. (Cf) Blow up of product reaction after Srs2 translocation.
(D) Quantification of the translocation assay presented in (C). Scale bars, 100 nm.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Model for Srs2 Action in the SDSA Pathway
(A) The presynaptic filament has invaded the donor molecule to form a D loop. The displaced strand is covered with RPA.
(B) Srs2 binds on the displaced strand and translocates with a 3′→5′ polarity.
(C) Srs2 liberates enough ssDNA to allow the binding of a new helicase molecule on the template strand (see text for more details).
(D) Thanks to its Rad51-stimulated helicase activity, Srs2 disassembles the D loop.
(E) The extended ssDNA anneals to the ssDNA on the other break end.
(F) Gap-filling DNA synthesis and ligation. Repair by SDSA leads to noncrossover product.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions