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. 2011 May;22(9):1599-607.
doi: 10.1091/mbc.E10-08-0691. Epub 2011 Mar 3.

The Shu Complex, Which Contains Rad51 Paralogues, Promotes DNA Repair Through Inhibition of the Srs2 Anti-Recombinase

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Free PMC article

The Shu Complex, Which Contains Rad51 Paralogues, Promotes DNA Repair Through Inhibition of the Srs2 Anti-Recombinase

Kara A Bernstein et al. Mol Biol Cell. .
Free PMC article

Abstract

The Shu complex, which contains RAD51 paralogues, is involved in the decision between homologous recombination and error-prone repair. We discovered a link to ribosomal DNA (rDNA) recombination when we found an interaction between one member of the Shu complex, SHU1, and UAF30, a component of the upstream activating factor complex (UAF), which regulates rDNA transcription. In the absence of Uaf30, rDNA copy number increases, and this increase depends on several functional subunits of the Shu complex. Furthermore, in the absence of Uaf30, we find that Shu1 and Srs2, an anti-recombinase DNA helicase with which the Shu complex physically interacts, act in the same pathway regulating rDNA recombination. In addition, Shu1 modulates Srs2 recruitment to both induced and spontaneous foci correlating with a decrease in Rad51 foci, demonstrating that the Shu complex is an important regulator of Srs2 activity. Last, we show that Shu1 regulation of Srs2 to double-strand breaks is not restricted to the rDNA, indicating a more general function for the Shu complex in the regulation of Srs2. We propose that the Shu complex shifts the balance of repair toward Rad51 filament stabilization by inhibiting the disassembly reaction of Srs2.

Figures

FIGURE 1:
FIGURE 1:
Overexpression of SHU1 causes an SDL interaction with deletion of UAF30, a gene that alters rDNA recombination in a FOB1-dependent manner. (A) Fivefold serial dilutions were plated onto selective media with increasing copper concentrations with the empty vector (pWJ1530) or the SHU1 overexpression plasmid (SHU1) transformed into WT or uaf30Δ. (B) Fluorescence microscopy of yeast cells containing Uaf30-YFP and Top1-CFP was analyzed for colocalization (Merge). (C) The frequency of rDNA recombinants (CANR, ade2) was measured in WT, uaf30Δ, fob1Δ, and uaf30Δ fob1Δ yeast strains, and they were plotted with SD. Note that rDNA recombination frequencies vary in uaf30Δ cells, likely due to the destabilization of the rDNA array.
FIGURE 2:
FIGURE 2:
Shu1 localizes to the nucleolus and affects rDNA recombination. (A) Yeast with Shu1-YFP-YFP and Nop1-CFP were analyzed by fluorescence microscopy for colocalization (Merge). (B) The frequency of rDNA recombinants (CANR, ade2) was measured in WT, shu1Δ, uaf30Δ, and uaf30Δ shu1Δ yeast strains, and they were plotted with SD. (C) The amounts of rDNA in WT, shu1Δ, uaf30Δ, and uaf30Δ shu1Δ strains were quantitated by analyzing the amount of rDNA resulting from restriction digest of total DNA, revealing a 9.1-kb fragment as described in Materials and Methods. SD are plotted. (D) WT and uaf30Δ strains were transformed with the empty vector (pWJ1530) or the SHU1 overexpression plasmid (pWJ1530-SHU1). Strains were grown in the presence of 100 μM copper (CuSO4), and the frequency of rDNA recombinants (CANR, ade2) was measured and the SE are plotted. Note that the recombination frequencies in uaf30Δ cells were different relative to (B), likely due to their growth in synthetic minimal medium, which was needed to maintain the plasmid.
FIGURE 3:
FIGURE 3:
Shu1 functions in the same pathway as Srs2 to suppress uaf30Δ rDNA recombination and alters Srs2 focus formation. (A) The frequency of rDNA recombination was measured in WT, shu1Δ, srs2Δ, shu1Δ srs2Δ, uaf30Δ, uaf30Δ srs2Δ, and uaf30Δ srs2Δ shu1Δ strains, and they were plotted with SD. Note the recombination frequency of the uaf30Δ shu1Δ strain was not conducted at the same time. (B) YFP-Srs2–expressing strains were analyzed for the percentage of spontaneous nuclear foci in WT, shu1Δ, uaf30Δ, and shu1Δ uaf30Δ cells. Images of Srs2 are shown with white arrowheads indicating foci. Each experiment was done in triplicate with a total of 400–500 cells analyzed. The graph shows the percentage of cells with foci along with the SE. (C) Cells expressing CFP-Rad51 were analyzed in WT and shu1Δ strains for the percentage of spontaneous nuclear foci. Each experiment was done in triplicate with a total of 150–200 cells analyzed with SE plotted. Note that the strains also contain a WT Rad51–complementing plasmid because CFP-Rad51 is not fully functional.
FIGURE 4:
FIGURE 4:
Shu1 inhibits Srs2 recruitment to DNA breaks. (A) An I-SceI cut site was integrated into the rDNA adjacent to a tandem array of Tet repressor–binding sites (224xtetO). Location of the rDNA break is revealed by expression of a TetI fused to mRFP. Rad52-CFP and YFP-Srs2 were monitored for their recruitment to rDNA breaks in WT and shu1Δ cells expressing a GAL-I-SceI plasmid. The results are quantitated in the graph with SE plotted. (B) An I-SceI cut site was integrated at the URA3 locus on chromosome V adjacent to a tandem array of the Tet repressor–binding sites (336xtetO). Rad52-CFP and YFP-Srs2 were monitored in WT and shu1Δ cells for recruitment to the cut site in strains expressing a GAL-I-SceI plasmid. The results are quantitated in the graph with SE plotted.
FIGURE 5:
FIGURE 5:
Rad51 filament formation is not inhibited in shu1Δ srs2Δ cells. WT, shu1Δ, srs2Δ, and shu1Δ srs2Δ cells were analyzed for the percentage of spontaneous CFP-Rad51 foci. Each experiment was done in triplicate with a total of 200 cells analyzed with SE plotted. Note that the strains also contain a WT Rad51–complementing plasmid because CFP-Rad51 is not fully functional. This configuration likely results in fewer Rad51 foci observed in srs2Δ cells than we previously reported (Burgess et al., 2009).
FIGURE 6:
FIGURE 6:
Model for repair of DNA breaks regulated by the Shu complex. After a DSB occurs in the DNA, the ends of the break site are resected and processed. Here we show a DSB, but this reaction can take place at any Rad51-mediated step. The Shu complex promotes Rad51 filament formation by inhibiting Srs2 recruitment to the break sites and preventing Srs2 inhibition of Rad51 filament formation. Alternatively, the Shu complex could directly promote Rad51 filament formation in a manner similar to other Rad51 paralogues (dashed line). After Rad51 filaments are formed (indicted by the beads on the single-strand tail), subsequent repair of the DNA lesion can occur via HR.

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