Multiple Regulation of Rad51-Mediated Homologous Recombination by Fission Yeast Fbh1

<div><p>Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCF^Fbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role <i>in vivo</i>, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange <i>in vitro</i> after strand exchange had been initiated.</p></div

Disruption of Rad51-ssDNA filaments is dependent on Fbh1 helicase/translocase activity.

<p>(A) Schematic diagram of the ssDNA-bead assay. After preparation of ssDNA beads, Rad51 is incubated to form Rad51-ssDNA filaments. After washing, Rad51-ssDNA filament was challenged with the Fbh1-Skp1 complex. After incubation, the supernatant (unbound) and bead (bound) fractions were analyzed by SDS-PAGE and visualized by CBB-G250 staining. (B) The effect of Fbh1-Skp1 dose on stability of the Rad51-ssDNA filament. Left, gel image of the assay; right, quantification of Fbh1-Skp1 dose effects. Images were captured, and the intensities of Rad51 protein bands were quantified. Summary of quantification is presented in the graph at right. The SDS-PAGE gel in the upper image was 9% (19∶1 acrylamide∶bisacrylamide); this condition was used to achieve better separation of CPK and Rad51 bands. The gel in the lower image was 9% (29∶1 acrylamide∶bisacrylamide). Each mean value and standard error (SE) was obtained from three independent experiments. (C) Fbh1, but not Skp1, displaces Rad51 from ssDNA. The ssDNA-bead assay was carried out using wild-type Fbh1, the Walker A mutant of Fbh1 (Fbh1^K301A), or Skp1 alone. Left, gel image of the assay. The two SDS-PAGE gels were 9% (19∶1 acrylamide∶bisacrylamide). Right, proportion of Rad51 recovered in the indicated fraction, relative to total amount of Rad51; each mean value and SE was obtained from three independent experiments.</p

Fbh1 inhibits the strand-exchange reaction at early steps, but stimulates it at the late steps.

<p>(A) Schematic of the three-strand exchange reactions. Fbh1 (90 nM) was added at the indicated step, (i)–(viii). (B) Gel image of three-strand exchange reactions. (C) Quantitation of results in panel B. Each mean value and SE was obtained from three independent experiments. (D) Three-strand exchange reactions were carried out with the indicated incubation times after addition of Swi5-Sfr1 (5–30 min). (E) Quantitation of results in panel D. Each mean value and SE was obtained from three independent experiments.</p

A model for regulation of HRR by Fbh1.

<p>After DSB formation, the ends of a DSB are nucleolytically processed to produce recombinogenic DNA with 3′ ss overhangs. Rad51 binds the ssDNA region, an interaction that is mediated by Rad52. Because the resulting Rad51 filament is not yet activated (‘premature’), it is prone to be disrupted by the ‘anti-recombinase’ activity of Fbh1. Swi5-Sfr1 protects the Rad51 filament by activating it. It remains unclear whether Rad55-Rad57 also protects the Rad51 filament. Previously, we proposed that both Rad55-Rad57 and Swi5-Sfr1 sub-pathways are involved in DSBR and synthesis-dependent strand annealing (SDSA), and that Rad55-Rad57 is required for second-end capture (SEC) to form the double-Holliday junction in DSBR <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004542#pgen.1004542-Akamatsu2" target="_blank">[8]</a>. In this study, the results of the single-DSBR assay suggest that Fbh1 suppresses crossover formation, most probably by inhibiting Rad55-Rad57-mediated SEC during DSBR. On the other hand, the <i>in vitro</i> data showing Fbh1 stimulation of branch migration in the strand-exchange reaction suggests that Fbh1 might have a pro-recombination role <i>in vivo</i>, probably by stimulating the SDSA pathway to generate a non-crossover repair product. This possibility needs to be addressed in future studies. Subsequently, Rad51 would be degraded by SCF^Fbh1-dependent ubiquitination to prevent re-activation, either after the reaction or during stationary phase.</p

The Swi5-Sfr1 activator complex blocks the Fbh1-mediated Rad51 dissociation from ssDNA.

<p>The ssDNA-bead assay was carried out as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004542#pgen-1004542-g001" target="_blank">Figure 1</a>. Intensity of Rad51 was quantified, and percentage reduction at each concentration of Swi5-Sfr1 was calculated as [(Rad51 amount on Beads in the presence of Fbh1)/(Rad51 amount on Beads in the absence of Fbh1)×100]. Upper panel shows the amount of Fbh1 in each lane. Each value and SE was obtained from three independent experiments.</p

Outcomes of HRR among survivors (% of total).

<p>The average frequencies of colonies produced by HRR (total HO-induced segregants = 100%) and standard errors were determined from the data sets in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004542#pgen.1004542.s009" target="_blank">Tables S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004542#pgen.1004542.s010" target="_blank">S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004542#pgen.1004542.s011" target="_blank">S3</a>.</p>a<p>Data are from Akamatsu et al. (2007) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004542#pgen.1004542-Akamatsu2" target="_blank">[8]</a>.</p>b<p>No colonies were obtained.</p><p>BIR, break induced replication; CO, crossover; GC, gene conversion; LTGC, long-tract gene conversion.</p><p>Outcomes of HRR among survivors (% of total).</p

Rad51 is ubiquitinated <i>in vitro</i> in an SCF^Fbh1-dependent manner.

<p>(A) Two kinds of E2 enzymes, Ubc4 and Ubc15, were used in the <i>in vitro</i> ubiquitination assays. After the reaction, the reaction mixture was analyzed by western blotting with anti-Rad51 antibody. Rad51 was ubiquitinated in a Ubc4- and SCF^Fbh1-dependent manner. (B) The helicase activity of Rad51 is dispensable for its ubiquitination, and ssDNA inhibited Rad51 ubiquitination. Both SCF^Fbh1WT and SCF^Fbh1-K301A promoted Rad51 ubiquitination, but no increase in ubiquitination was observed in the absence of SCF. (C) <i>In vitro</i> ubiquitination assay containing Swi5-Sfr1. Swi5-Sfr1 inhibited Rad51 ubiquitination in a dose-dependent manner. (D) Comparison of Rad51 and Rad52 protein levels in log and stationary phases. Rad51 protein was degraded in stationary phase in wild-type cells, but this degradation was blocked by <i>fbh1</i> mutations; consequently, Rad51 protein levels were higher in <i>fbh1</i> mutants than in the wild-type strain.</p