Illustration of error-free repair pathways of a DSB. See text for details.

Identification of nicked and gapped circular DNA products by strand-specific labeling of dsDNA substrate. (A) The ‘+' strand of the linear dsDNA substrate (marked by filled star) is partially complementary to the ssDNA(SK−), and ‘−' strand (marked by open star) is fully complementary to ssDNA(SK+). DNA strand exchange followed by annealing of the displaced DNA strand produces nicked and gapped circular products that receive ‘−' and ‘+' strands from the dsDNA, respectively. (B, C) Two-step DNA strand exchange and annealing (same reactions as shown in Figure 2C, lane 5) were performed except that the dsDNA substrates were labeled with ³²P at ‘+' strand (B) or ‘−' strand (C). Gels were visualized by ethidium bromide staining (lanes 1) and then by phosphorimaging (lanes 2).

RPA(rfa1-t11) does not support DNA annealing by Rad52. (A) Lanes 1 and 2 show DNA strand exchange reaction between the partially homologous substrates with Rad51 only (lane 1) or with Rad51 and RPA(rfa1-t11) (lane 2). Lanes 3 and 4 show two-step DNA strand exchange and annealing with RPA(rfa1-t11). DNA strand exchange between the partially homologous substrates was performed with Rad51 and RPA(rfa1-t11), and then followed by an incubation (60 min) with RPA(rfa1-t11)–ssDNA(SK+) complex (lane 3) or with both RPA(rfa1-t11)–ssDNA(SK+) complex and Rad52 (lanes 4). (B) DNA annealing was analyzed using heat-denatured pBluescript SK− dsDNA in the identical buffer conditions to the two-step DNA strand exchange and annealing. DNA (10 μM) was preincubated with 0.5 μM of either wild-type RPA or RPA(rfa1-t11), then with (+) or without (–) 1.59 μM of Rad52 for the indicated periods, and the products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. Linear ssDNA substrate (ss), dsDNA product (ds), and loading well (well) are indicated. (C) Same experiments as (B) were performed, except that (C) contained no KCl in the reaction buffer. (D–E) Percentages of DNA annealing are calculated from duplicated experiments of panel B (D), and panel C (E). For both (D) and (E), curves represent the products formations with Rad52 and either wild-type RPA (open circle with solid line) or RPA(rfa1-t11) (filled triangle with dashed line), only with wild-type RPA (filled circle with solid line), or only with RPA(rfa1-t11) (filled square with dashed line). Error bars represent standard deviations.

Coordinated DNA strand exchange and annealing. (A) Illustration of the reaction scheme. ssDNA(SK−) was first incubated with RPA, followed by Rad52 and Rad51. After 10 min, dsDNA was added to start DNA strand exchange (T=0 min). At T=10 min, the RPA–ssDNA(SK+) complex was added to initiate annealing of the second ssDNA. (B) Reaction illustrated in (A) was performed without the second ssDNA, and time course was taken after addition of dsDNA, and products were analyzed by agarose gel electrophoresis. (C) Reaction illustrated in (A) was performed with RPA–ssDNA(SK+) complex that was added at T=10 min. (D) Coordinated reactions (the same reaction as lane 5 in (C)) were carried out in the presence of different concentrations of Rad52. (E) Amounts of joint molecules in (B) (filled square) and (C) (open circle), and the sum of nicked and gapped circular DNA products in (C) (filled circle) were expressed as the percentage to the total amount of DNA. (F) Joint molecule (open circle) and the sum of nicked and gapped circular DNA products (filled circle) in (D) were expressed as the percentage to the total amount of DNA. (G) Model for action of RPA, Rad52, and Rad51 in DSB repair. See text for details.

Rad52 mediates annealing of the displaced DNA strand that is produced by DNA strand exchange by Rad51. (A) Illustration of two-step DNA strand exchange and annealing. See text for details. (B) Illustration of DNA strand exchange between homologous ssDNA and dsDNA. Reaction produces joint molecules, nicked circular dsDNA, and linear ssDNA. (C) Lanes 1 and 2: DNA strand exchange between ssDNA and homologous dsDNA (illustrated in (B)) was performed in the absence (lane 1) or the presence (lane 2) of Rad51 and RPA. Lanes 3–7: Two-step DNA strand exchange and annealing (illustrated in (A)). DNA strand exchange between partially homologous substrates was performed by Rad51 and RPA (lane 4), and the reaction mixture was subsequently incubated for 60 min (Step 2 in panel A) with RPA–ssDNA(SK+) complex and Rad52 (lane 5), with only RPA–ssDNA(SK+) (lane 6), or with only Rad52 (lane 7). Lane 3 is no-protein control. Joint molecule (JM), gapped circle (GC), nicked circle (NC), linear double-strand (L), and single-stranded (ss) DNA are indicated. Besides the molecules mentioned in the text, the ssDNA of helper phage (VCSM13, indicated as ‘H') was present in the SK− ssDNA preparation. Another band, which was indicated by an asterisk, was produced most evidently in lane 5. This is a product of partial annealing between SK− and SK+ circular ssDNA molecules, as this band also appeared when these ssDNA molecules were incubated with Rad52 without dsDNA (data not shown). This band is also visible in other gels (Figures 4, 5, 6, and 7), and indicated by asterisks. Linear ssDNA that was produced in the reaction comigrated with the circular ssDNA. (D) Same experiment as lane 5 of (C) was performed in the presence of various amount of Rad52, and joint molecule (open circle) and the sum of nicked and gapped circular DNA products (open triangle) were expressed as the percentage to the total amount of DNA.

Rad52-mediated DNA annealing promotes quick separation of joint molecule into circular products. (A–C) Same reaction as shown in Figure 2C, lane 5 was repeated in a larger volume, and aliquots were withdrawn to stop the reaction at the indicated times after addition of Rad52. As a negative control, same reaction as (B) was performed without Rad52 (C). Products were analyzed by agarose gel electrophoresis and ethidium bromide staining. The reactions for lanes 1 and 7 were stopped before addition of Rad52. (D) Amount of nicked (open circle) and gapped (filled square) circular DNA products and joint molecules (open triangle) were quantified from (B), and amount of gapped circle was quantified from (C) (filled circle), and expressed as the percentage of the total amount of DNA. (E) Possible intermediates of the reaction.

Species-specific interactions between ssDNA-binding protein and recombination mediator are involved in the second-ssDNA capture. DNA strand exchange between partially homologous substrates (Step 1) was performed under the standard conditions except that either E. coli SSB protein (58.8 pmoles), RPA (20.6 pmoles), or no ssDNA-binding protein was used as indicated. Then, annealing of the displaced DNA strand (Step 2, lanes 3–8) was performed using ssDNA(SK+) complexed with either SSB or RPA, and the indicated amount of Rad52 or RecO.