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. 2016 Jun 7;15(10):2118-2126.
doi: 10.1016/j.celrep.2016.05.007. Epub 2016 May 26.

Promotion of RAD51-Mediated Homologous DNA Pairing by the RAD51AP1-UAF1 Complex

Fengshan Liang  1 , Simonne Longerich  1 , Adam S Miller  1 , Caroline Tang  2 , Olga Buzovetsky  1 , Yong Xiong  1 , David G Maranon  3 , Claudia Wiese  3 , Gary M Kupfer  4 , Patrick Sung  5
Affiliations

Promotion of RAD51-Mediated Homologous DNA Pairing by the RAD51AP1-UAF1 Complex

Fengshan Liang et al. Cell Rep. .

Abstract

The UAF1-USP1 complex deubiquitinates FANCD2 during execution of the Fanconi anemia DNA damage response pathway. As such, UAF1 depletion results in persistent FANCD2 ubiquitination and DNA damage hypersensitivity. UAF1-deficient cells are also impaired for DNA repair by homologous recombination. Herein, we show that UAF1 binds DNA and forms a dimeric complex with RAD51AP1, an accessory factor of the RAD51 recombinase, and a trimeric complex with RAD51 through RAD51AP1. Two small ubiquitin-like modifier (SUMO)-like domains in UAF1 and a SUMO-interacting motif in RAD51AP1 mediate complex formation. Importantly, UAF1 enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1 but independent of USP1. Mechanistically, RAD51AP1-UAF1 co-operates with RAD51 to assemble the synaptic complex, a critical nucleoprotein intermediate in homologous recombination, and cellular studies reveal the biological significance of the RAD51AP1-UAF1 protein complex. Our findings provide insights into an apparently USP1-independent role of UAF1 in genome maintenance.

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Figures

Figure 1
Figure 1. RAD51AP1-UAF1 complex formation via the SIM and SLD1-SLD2 domains in these proteins
(A) Strep II-tagged UAF1, UAF1-436X, MBP-tagged UAF1-SLD1, UAF1-SLD2, and UAF1-SLD1/SLD2 (SLD1/2) were incubated with GST-tagged RAD51AP1, and protein complexes were captured on glutathione resin and analyzed by SDS-PAGE. S, supernatant containing unbound proteins; W, wash; E, SDS eluate of the glutathione resin. (B) Alignment of UAF1-SLD1 against SUMO2 is shown. The asterisk highlights the K459 and K33 residues in UAF1 and SUMO2, respectively. Arrow and helix represent beta sheet and alpha helix, respectively. (C) GST-tagged RAD51AP1 was incubated with Strep II-tagged UAF1 (WT) or the indicated UAF1 mutant, and protein complexes were captured with glutathione resin. Analysis was as in (A). (D) Sequence analysis reveals a SIM between amino acid residues 137-142 in RAD51AP1. The asterisks highlight the residues targeted for mutagenesis. Hs, Homo sapiens; Mm, Mus musculus; Bt, Bos taurus; Rn, Rattus norvegicus; Pt, Pan troglodytes. (E) GST-tagged RAD51AP1 (WT) or the indicated RAD51AP1 mutant was incubated with Strep II-tagged UAF1, and protein complexes were captured with glutathione resin. Analysis was as above. (F) Schematic highlighting the RAD51AP1-UAF1 interaction domains. See also Figure S1.
Figure 2
Figure 2. Association of UAF1 with RAD51 via RAD51AP1 and DNA-binding activity in UAF1
(A) Strep II-tagged UAF1 (WT) or UAF1-EEA was incubated with RAD51AP1, RAD51AP1-LI2A, and RAD51 alone or in combination. Protein complexes were captured on Strep-Tactin resin and the different fractions were analyzed as in Figure 1A. MBP-tagged UAF1 SLD1-SLD2 (SLD1/2) was similarly incubated with RAD51AP1 and RAD51, and amylose resin was used to capture the trimeric protein complex. A control experiment confirmed that RAD51AP1-RAD51 does not bind amylose resin nonspecifically (data not shown). (B) Strep II-tagged UAF1 was incubated with RAD51AP1, yeast Rad51 or their combination, and Strep-Tactin pulldown was carried out as in (A). (C) Strep II-tagged UAF1-FL or UAF1-436X was incubated with radiolabeled 80-merss DNA. The mobility shift of the DNA was analyzed. Treatment with SDS and proteinase K (SDS+PK) released the DNA from nucleoprotein complexes. The data were quantified and plotted. The error bars represent mean values ± S.D. of data from three independent experiments. (D) The ability of UAF1 and UAF1-436X to bind radiolabeled dsDNA was analyzed as in (C). See also Figure S2.
Figure 3
Figure 3. Synergistic action of RAD51AP1 and UAF1 in RAD51-mediated D-loop formation
(A) Schematic of the D-loop assay. (B) RAD51, RAD51AP1, Strep II-tagged UAF1, and the indicated combinations of these proteins were tested for the ability to form D-loops. The percentages of D-loop product from three independent experiments are shown as the mean ± S.D. (C) The K459E and EEA mutants of UAF1 were tested with RAD51AP1 in the D-loop reaction. Data analysis was as in (B). (D) The IV2A and LI2A mutants of RAD51AP1 were tested with UAF1 in the D-loop reaction. See also Figure S3.
Figure 4
Figure 4. Duplex DNA capture and synaptic complex assembly by RAD51-RAD51AP1-UAF1
(A) Schematic of the duplex capture assay. (B) RAD51AP1, Strep II-tagged UAF1, or the combination of these proteins was incubated with the RAD51 presynaptic filament, and the ability to capture dsDNA was analyzed. The percentages of captured dsDNA are shown. The error bars represent mean values ± S.D. of data from three independent experiments. (C) Schematic of synaptic complex assembly as assayed by protection against SspI digestion. (D) RAD51AP1, Strep II-tagged UAF1, or their combination was incubated with the RAD51 presynaptic filament, and the protection of dsDNA against SspI digestion was analyzed. Lane 1 was the control without SspI treatment. The protected DNA was quantified and plotted. Error bars are mean ± S.D. of three independent experiments. See also Figure S4.
Figure 5
Figure 5. Role of the RAD51AP1-UAF1 complex in DNA damage repair and HR
(A) Extracts from HeLa cells expressing wild type (WT) or the mutant form (K459E or EEA) of FLAG-tagged UAF1 were subject to co-immunoprecipitation analysis with anti-FLAG M2 agarose resin. Proteins were revealed by Western blotting. (B) Protein levels in HeLa cells with constitutive shRNA-mediated knockdown of UAF1 and stably expressing either wild type or the indicated mutant form of UAF1 were determined by Western blotting. Cells without shUAF1 were included as control. Ku86 was used as the loading control. (C) HeLa cells described in (B) were treated with MMC, CPT or olaparib, and cell numbers were determined after incubation at 37°C for 5 days and normalized to the untreated control. The percentages of surviving cells are shown as the mean ± S.D. from three independent experiments. (D) U2OS-DR-GFP cells with constitutive shUAF1-mediated knockdown of UAF1 and stably expressing either wild type or a mutant form of UAF1 were transfected with HA-tagged I-SceI plasmid and processed for flow cytometric analysis of GFP. The protein levels are shown in the left panel. The repair efficiency was scored as the percentage of GFP-positive cells. C, Control cells without I-SceI; 1, shRNA control cells; 2, shUAF1 cells; 3,shUAF1 cells with UAF1; 4, shUAF1 cells with UAF1-EEA; 5, shUAF1 cells with UAF1-K459E. Error bars indicate S.E.M. ** indicates P < 0.01, unpaired t-test. (E) Model for the roles of UAF1 in the FA pathway and HR. UAF1 functions as the substrate targeting subunit of the DUB enzyme central to the FA pathway and also as an important cofactor of RAD51-RAD51AP1 to facilitate the assembly of the synaptic complex during HR. See also Figure S5.
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