1. Volume 6, Issue 3, Pages 563-572 (September 2000)
Superhelicity-Driven Homologous DNA Pairing by Yeast Recombination Factors Rad51 and Rad54 
Stephen Van Komen, Galina Petukhova, Stefan Sigurdsson, Sabrina Stratton, Patrick Sung 
Molecular Cell 
Volume 6, Issue 3, Pages (September 2000)
DOI: /S (00)

2. Figure 1 Effect of Superhelicity on D Loop Formation
(A) Schematic of the D loop reaction. Linear ssDNA is paired with a covalently closed duplex, such as a negatively supercoiled species as shown, to yield the D loop. A variety of covalently closed duplex substrates differing in their topological state were employed in the present study (see below).
(B) Superhelicity and D loop formation. In (I) through (V), a preassembled nucleoprotein complex of Rad51-RPA-ssDNA was mixed with Rad54 and duplex DNA substrates that were positively supercoiled (+SC in [I]), relaxed (Rl in [II]), and with σ values of −0.021 (III), −0.039 (IV), and −0.076 (V), as indicated. The reaction mixtures were analyzed in agarose gels containing 10 μM ethidium bromide to effect the separation of D loop species from other DNA species (nicked circular duplex and linear duplex, which are not marked). At later time points, more complex D loop species appeared, which likely corresponded to multiple molecules of ssDNA and dsDNA paired together. The linear single-strand is designated as ss.
(C) The results in (B) are plotted.
(D) Results from D loop reactions in which RecA-SSB-ssDNA complex was reacted with duplex substrates of the indicated topological state are plotted.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2
DNA Remodeling by Rad54 as Revealed by Topoisomerase Treatment
(A) Increasing concentrations of Rad54 protein (0.045, 0.09, 0.23, 0.45, 0.9, and 1.8 μM in lanes 2 to 7 and lanes 9 to 14, respectively) were incubated with relaxed DNA and with either calf thymus topoisomerase I (lanes 2 to 7) or E. coli topoisomerase I (lanes 9 to 14), as indicated. In lanes 1 and 8, DNA was incubated in buffer with topoisomerase but no Rad54 protein. The novel DNA species generated by the combination of Rad54 and topoisomerase I is designated as Form OW, and the heterogeneous novel DNA species generated by the combination of Rad54 and calf thymus topoisomerase I are collectively designated as Form U. The DNA concentration was 18.5 μM base pairs, and all the reactions were incubated at 37°C for 10 min.
(B) Formation of Form OW DNA requires ATP hydrolysis. Rad54 (0.3 μM) was incubated with relaxed DNA and E. coli topoisomerase I in the presence of ATP (lane 2), ADP (lane 5), ATP-γ-S (lane 6), or in the absence of a nucleotide (lane 4), as indicated. The reaction in lane 1 contained relaxed DNA without Rad54, and in lane 3, relaxed DNA was incubated with Rad54 and ATP without topoisomerase. In lanes 7 and 8, rad54 K341A (K/A) and rad54 K341R (K/R) mutant proteins, 0.3 μM each, were incubated with relaxed DNA, E. coli topoisomerase I, and ATP, as indicated. The DNA concentration was 18.5 μM base pairs, and the reactions were carried out at 37°C for 10 min.
(C) Two-dimensional gel analysis of Form OW DNA. In (I) and (II), negatively supercoiled φX DNA isolated from cells σ = −0.06 without (I) or with (II) prior treatment with E. coli topoisomerase I was subject to two-dimensional gel analysis. In (III) and (IV), a mixture of negatively supercoiled φX DNA and purified Form OW DNA without (III) or with (IV) prior treatment with E. coli topoisomerase I was subject to two-dimensional gel analysis. Note in (IV) that the negatively supercoiled DNA, but not Form OW DNA, was relaxed by E. coli topoisomerase I. OW, Form OW DNA; SC, negatively supercoiled DNA; Rl, relaxed DNA; and nc, nicked circular DNA. In these gel analyses, the first dimension was conducted in the absence of chloroquine (−CQ) and the second dimension in the presence of chloroquine (+CQ).
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3
Effect of Rad51 on the Rad54 DNA Supercoiling and ATPase Activities
(A) Stimulation of DNA supercoiling by Rad51 and Rad51-RPA-ssDNA complex. Rad54, at 22.5 nM (lanes 2 to 8), was incubated with relaxed φX DNA (18.5 μM base pairs, lanes 1 to 8), E. coli topoisomerase I (lanes 1 to 8), and increasing amounts of Rad51 (120, 240, and 360 nM in lanes 3 to 5) or nucleoprotein complexes of Rad51-RPA-ssDNA (Rad51-fil) containing a constant amount of RPA (2 μM) and pBluescript ssDNA (30 μM nucleotides) but an increasing level of Rad51 (60, 120, and 240 nM in lanes 6 to 8) at 23°C for 10 min.
(B) RecA does not stimulate DNA supercoiling. Relaxed DNA (lanes 1 to 12) was incubated with E. coli topoisomerase I (lanes 1 to 12), Rad54 (lanes 2, 4, 6, 7, 9, 10, and 12), Rad51 (lanes 3 and 4), Rad51-RPA-ssDNA complex (lanes 8 and 9), RecA (lanes 5 and 6), and RecA-SSB-ssDNA complex (lanes 11 and 12), all in the presence of ATP. The concentrations of the reaction components were Rad54, 90 nM; Rad51, 120 nM; RPA, 2 μM; pBluescript ssDNA, 30 μM nucleotides; relaxed φX DNA, 18.5 μM base pairs; RecA, 1 μM; and SSB, 6 μM. The reaction mixtures were incubated at 23°C for 10 min.
(C) Dependence of DNA supercoiling on ATP hydrolysis. Relaxed DNA (lanes 1 to 6) was incubated with E. coli topoisomerase I (lanes 1 to 6), Rad54 (lanes 1 to 6), and Rad51 (lanes 2 and 5) or a nucleoprotein complex of Rad51, RPA, and ssDNA (Rad51-fil; lanes 3 and 6) in the presence of ATP (lanes 1 to 3) or ATP-γ-S (lanes 4 to 6), as indicated. Likewise, relaxed DNA (lanes 7 to 12) was incubated with E. coli topoisomerase I (lanes 7 to 12), rad54 K341R (K/R; lanes 7 to 9), and rad54 K341A (K/A; lanes 10 to 12) and either Rad51 (lanes 8 and 11) or the Rad51-RPA-ssDNA complex (Rad51-fil; lanes 9 and 12), all in the presence of ATP. The concentrations of the reaction components were Rad54, rad54 K341A, and rad54 K341R proteins, 90 nM; relaxed φX DNA, 18.5 μM base pairs; Rad51, 120 nM; RPA, 2 μM; and pBluescript ssDNA, 30 μM nucleotides. The reaction mixtures were incubated at 23°C for 10 min.
(D) Rad51 and Rad51-RPA-ssDNA complex stimulate Rad54 ATPase activity. Rad54, at 50 nM, was incubated with radiolabeled ATP and relaxed φX DNA without other protein (open triangles), with Rad51 (closed circles), or with Rad51-RPA-ssDNA complex (open circles) at 23°C.
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4 DNA Supercoiling Leads to DNA Strand Opening
(A) Rad54 renders topologically relaxed DNA sensitive to P1 nuclease. Relaxed DNA (18.5 μM base pairs, lanes 1 to 8) was incubated at 23°C for 10 min with P1 nuclease and increasing concentrations of Rad54 protein (90, 250, and 500 nM in lanes 4 to 6, respectively) in the presence of ATP. In lane 3, Rad54 (500 nM) was incubated with relaxed DNA in the presence of ATP but without P1, and in lanes 7 and 8, Rad54 (500 nM), relaxed DNA, P1 nuclease were incubated in the absence of ATP or with ATP-γ-S, as indicated.
(B) Rad51 enhances P1 sensitivity. In (I), relaxed φX DNA (18.5 μM base pairs in lanes 1 to 10) was incubated with P1 nuclease (lanes 1 to 10), Rad54, (50 nM; lanes 2 to 10), and increasing amounts of Rad51 (60, 120, 240, and 360 nM in lanes 3 to 6, respectively) or nucleoprotein complexes containing pBluescript ssDNA (30 μM nucleotides), RPA (2 μM), and increasing amounts of Rad51 (60, 120, 240 and 360 nM in lanes 7 to 10, respectively) at 23°C for 10 min. In lane 1, relaxed DNA and P1 nuclease were incubated in the absence of Rad51 and Rad54. In (II), relaxed φX DNA (18.5 μM base pairs in lanes 1 to 6) was incubated with P1 nuclease (lanes 1 to 6), Rad54 (50 nM in lanes 2, 4, and 6), and Rad51 (360 nM in lanes 3 and 4) or a nucleoprotein complex of Rad51-RPA-ssDNA (Rad51-fil, consisting of 360 nM Rad51, 2 μM RPA, and 30 μM pBluescript ssDNA; lanes 5 and 6), as described in (I).
(C) RecA does not promote DNA strand opening. Relaxed φX DNA (18.5 μM base pairs; lanes 1 to 11), P1 nuclease (lanes 1 to 11), Rad54 (50 nM in lanes 6 to 11), and Rad51 (360 nM, lane 10), RecA (525 nM in lanes 2 and 6, and 2.6 μM in lanes 3 and 7), or nucleoprotein complexes of RecA-SSB-ssDNA (RecA-fil; lanes 4, 5, 8, and 9) consisting of a fixed amount of SSB (6 μM), pBluescript ssDNA (30 μM nucleotides), but an increasing level of RecA (525 nM in lanes 4 and 8, and 2.6 μM in lanes 5 and 9). The analyses in (A) through (C) were carried out in 0.85% agarose gels containing 10 μM ethidium bromide.
Molecular Cell 2000 6, DOI: ( /S (00) )

6. Figure 5
DNA Binding by Rad51 Is Required for DNA Supercoiling and D Loop Formation
(A) DNA binding ability of wild-type Rad51 and mutant rad51 proteins. Rad51 (1, 2, 4, and 6 μM in lanes 2 to 5, and 6 μM in lane 14), rad51 K191R (1, 2, 4, and 6 μM in lanes 6 to 9, and 6 μM in lane 15), and rad51 K191A (1, 2, 4, and 6 μM in lanes 10 to 13, and 6 μM in lane 16) were incubated with a mixture of φX viral (+) strand (30 μM nucleotides) and linear duplex (20 μM base pairs), either in the presence (lanes 1 to 13) or absence of ATP (14 to 16), for 10 min at 23°C. The reaction mixtures were run in a 0.9% agarose gel to detect the mobility shift of the DNA substrates.
(B) rad51 K191R but not rad51 K191A forms D loop with Rad54. Time courses of D loop formation (I) with Rad51 (1.5 μM), rad51 K191A (1.5 μM), and rad51 K191R (1.5 μM) using φX linear ssDNA (ss) and negatively supercoiled DNA (sc) with σ = −0.06 isolated from cells as substrates. The details are given in the Experimental Procedures. The datapoints from image analysis of the gels are plotted (II).
(C) rad51 K191R but not rad51 K191A cooperates with Rad54 in supercoiling DNA. The relaxed φX DNA substrate (18.5 μM) was incubated with Rad54 (90 nM in lanes 2 to 9), E. coli topoisomerase I (lanes 1 to 9), Rad51 (120 nM, lane 3), rad51 K191R (120, 240, and 360 nM in lanes 4, 5, and 6, respectively), and rad51 K191A (120, 240, and 360 nM in lanes 7, 8, and 9, respectively) for 10 min at 23°C, as indicated.
(D) Enhancement of DNA strand opening by rad51 K191R but not rad51 K191A. Relaxed φX DNA (18.5 μM base pairs) was incubated with P1 nuclease and the following components at 23°C for 10 min before gel analysis: Rad54 (90 nM in lanes 2 to 8), Rad51 (WT; 240 and 360 nM in lanes 3 and 4, respectively), rad51 K191R (K/R; 360 and 480 nM in lanes 5 and 6, respectively), and rad51 K191A (K/A; 360 and 480 nM in lanes 7 and 8, respectively), as indicated. The analyses in (D) were carried out in 0.85% agarose gels containing 10 μM ethidium bromide.
Molecular Cell 2000 6, DOI: ( /S (00) )