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Fig. 1. Generic model for assembly of recombinase on ssb-coated ssDNA. (A) Tracts of ssDNA form because of resection at DSB sites or stalling of polymerase. (B) ssb assembles into oligomeric filaments on tracts of ssDNA, removing secondary structures. (C) Mediator protein binds to ssb-coated DNA, causing a local remodeling of the ssb filament. (D) Recombinase initiates filament formation at sites of mediator-ssb-ssDNA. Recombinase filaments then elongate displacing ssb. (E) The elongated recombinase filament searches for homologous sequences. (F) Strand exchange occurs. The outgoing ssDNA strand is bound by ssb. filaments are initiated, interactions that occur during recombinase filament elongation displace ssb from DNA. Elongation of recombinase filaments is blocked by DNA secondary structures, whereas assembly of ssb filaments is not. As a result of these properties, adding ssb to reactions after recombinase allows recombinase the opportunity to initiate filaments on ssDNA before ssb is added and then extend past sites of secondary structure with the help of ssb. The recombinase filaments formed as a result of ssb-recombinase interactions are more efficient in strand exchange reactions than filaments formed by recombinase alone because of persisting secondary structures in ssDNA. RPA Assembles Before Rad51 After Radiation Treatment. Immuno-staining techniques can detect assembly of recombination proteins into subnuclear staining foci (23–32). Several lines of evidence suggest that these foci represent protein oligomers assembled at sites of DSBs and possibly daughter strand gaps as well. Double staining shows that multiple proteins required for recombination assemble at the same sites during recombination. Such experiments in yeast and mammalian cells showed that RPA colocalizes with Rad51 (29, 31–33). An important question raised by the biochemical interaction of ssb and recombinase is: Does ssb assemble before or after recombinase in vivo? The high efficiency of binding to ssDNA and the abundance of ssb predict that ssb will assemble more rapidly than recombinases on tracts of ssDNA. However, the one previous experiment that addressed the question of the relative timing of assembly of RPA and Rad51 during recombination found that Rad51 foci assembled first, with colocalizing RPA foci appearing at a later stage during meiosis in mouse spermatocytes (33). To determine the relative timing of appearance of RPA and 
Fig. 2. RPA assembles before Rad51 after γ-irradiation of G2 arrested S. cerevisiae spheroplasts. A diploid yeast strain (NKY1314, SK-1 strain background) was grown in rich medium and arrested in G2/M by treatment with nocodazole. Cells were spheroplasted by zymolyase treatment, irradiated to a dose of 50 krad with a 60Co source, and resuspended in osmotically stabilized growth medium containing nocodazole. At the times indicated, culture aliquots were processed to obtain surface spread nuclei. Spread nuclei were indirectly immunostained with polyclonal guinea pig anti-Rad51 (green) and rabbit anti-RPA70 (red), counter strained with a DNA-specific dye (DAPI), and examined by epifluorescence microscopy. The number of foci per nucleus was determined by visual examination of digital images. Details of the method have been published (29, 59). (A) Representative nuclei from the times indicated. Overlapping signals are yellow in these pseudocolored images, (B) Average number of foci detected per nucleus at the times indicated. Rad51 foci after induction of damage by γ-rays, a log phase culture of diploid cells was arrested in G2/M with the microtubule inhibitor nocodazole. Cells were arrested at the G2/M phase of the cell cycle to avoid detection of the RPA foci that form in undamaged cells as a normal part of S phase (29). In addition to being arrested in G2/M, cells were treated with zymolyase to remove the cell wall, which allowed rapid processing of samples. After treatment of arrested cells with 50 krad, the culture was incubated at 30°C for various times and spread nuclei prepared for immunostaining. Fifty unselected surface-spread nuclei were examined from each sample. The data are shown in Fig. 2. Induction of RPA focus positive nuclei was detected immediately after treatment, i.e., at the time point designated t= 0 h. The RPA foci seen in these nuclei depend on irradiation, because they did not appear in an untreated control. Induction of Rad51 foci was not detected until t=0.25 h. The average number of induced RPA-containing foci increased to a maximum of 33/nucleus at 2 h. Rad51 containing foci also peaked at 2 h (18/nucleus). At all times, the majority of Rad51-containing foci (67–83%) also stained with RPA. The subset of Rad51 foci that did not contain detectable amounts of RPA could result from assembly of Rad51 at sites devoid of RPA. Alternatively, they could result from displacement of RPA, epitope masking of
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