Scaffolding protein SPIDR/KIAA0146 connects the Bloom syndrome helicase with homologous recombination repair Li Wan1, Jinhua Han1, Ting Liu1, Shunli Dong, Feng Xie, Hongxia Chen, and Jun Huang2 Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China Edited by James E. Cleaver, University of California, San Francisco, CA, and approved February 26, 2013 (received for review December 1, 2012) The Bloom syndrome gene product, BLM, is a member of the highly of the SDSA pathway (6, 7). The ability of BLM to yield non- conserved RecQ family. An emerging concept is the BLM helicase crossover products is thought to play a critical role in the avoidance collaborates with the homologous recombination (HR) machinery to of chromosomal rearrangements during the homolog-directed re- help avoid undesirable HR events and to achieve a high degree of pair of chromosomal lesions. As a result, cells defective for BLM fidelity during the HR reaction. However, exactly how such coordina- exhibit elevated rates of sister chromatid exchange (SCE) (19–21). tion occurs in vivo is poorly understood. Here, we identified a protein Upon the occurrence of DNA damage, BLM is able to form termed SPIDR (scaffolding protein involved in DNA repair) as the link discrete foci, where it colocalizes with other DNA repair proteins between BLM and the HR machinery. SPIDR independently interacts (22, 23). However, mechanistically how BLM is recruited to sites with BLM and RAD51 and promotes the formation of a BLM/RAD51- of DNA damage and how it collaborates with other proteins to containing complex of biological importance. Consistent with its role mediate recombination repair remain largely unexplored. In this as a scaffolding protein for the assembly of BLM and RAD51 foci, cells study, we used an affinity purification approach to isolate BLM- depleted of SPIDR show increased rate of sister chromatid exchange containing complex and identified a unique scaffolding protein and defects in HR. Moreover, SPIDR depletion leads to genome in- KIAA0146, which we refer to as SPIDR (scaffolding protein in- stability and causes hypersensitivity to DNA damaging agents. We volved in DNA repair). We demonstrate that SPIDR directly propose that, through providing a scaffold for the cooperation of interacts with BLM and this interaction is required to target BLM CELL BIOLOGY BLM and RAD51 in a multifunctional DNA-processing complex, SPIDR to sites of DNA damage. Consistent with a role in BLM-mediated not only regulates the efficiency of HR, but also dictates the specific processes, cells depleted of SPIDR display an increased level HR pathway. of sister chromatid exchange. Moreover, we found that SPIDR promotes HR through a direct interaction with the recombinase DNA double-strand breaks | noncrossover | double Holliday junction RAD51. Finally, we show SPIDR promotes the formation of a BLM/RAD51-containing complex of biological importance. We omologous recombination (HR) repair allows precise repair therefore propose that, through licensing key cellular biochemical Hof DNA double-strand breaks and is critical for restarting properties of BLM and RAD51, SPIDR not only regulates the stalled or collapsed DNA replication forks (1–4). The process efficiency of HR but also dictates the specificHRpathway. of HR in eukaryotic cells requires several proteins, the central player of which is the RAD51 recombinase (1–4). HR repair is Results initiated by nuclease-mediated DNA end resection to generate Identification of SPIDR as a Unique BLM-Binding Partner. To search 3′-single-stranded DNA (ssDNA) tails that are initially coated by for previously undetected proteins present in BLM-containing the replication protein A (RPA) complex (1–4). In a subsequent complex, we performed tandem affinity purification (TAP) using step, recombination mediator proteins catalyze the replacement 293T cells stably expressing streptavidin-flag-S protein (SFB)- of RPA with RAD51, resulting in the formation of ssDNA-RAD51 tagged wild-type BLM for the identification of BLM-interacting filament (1–4). The ssDNA-RAD51 filament then catalyzes strand proteins. Mass spectrometry analysis revealed several known invasion into homologous duplex DNA, leading to the formation of BLM-associated proteins, including TopoIIIα, RMI1/2, Rif1, a displacement loop (D-loop) (1–4). After DNA synthesis primed and FANCM (Fig. 1A). Interestingly, we also repeatedly iden- by the invading strand, the repair can bifurcate into two alternative tified a previously uncharacterized BLM-binding protein as subpathways referred to as synthesis-dependent strand annealing KIAA0146 (Fig. 1A). To ensure that KIAA0146 indeed associates (SDSA) and double-strand break (DSB) repair (5–9). In SDSA, the with BLM, we performed reverse TAP using a cell line stably extended D-loop can be dissolved by DNA helicases and the newly expressing tagged KIAA0146, and identified BLM, TopoIIIα, synthesized strand is annealed to the ssDNA tail on the other break Rif1, RMI1/2, and FANCM as major KIAA0146-associated end, which is followed by gap-filling DNA synthesis and ligation proteins (Fig. 1B). These data strongly suggest that KIAA0146 (5–9). The repair products from SDSA are always noncrossover is a bone fide BLM-binding protein. The KIAA0146 gene enc- (5–9). In DSB repair, the second DSB end is captured to form an odes a deduced polypeptide of 915 amino acids with a predicted intermediate with two Holliday junctions, called double Holliday molecular mass of 105 kDa. Database searches did not identify junction (dHJ) (5–9). dHJ can be processed to yield crossover or noncrossover recombination products (5–9). There is mounting evidence that DNA repair mechanisms in mitotically proliferating Author contributions: L.W., J. Han, T.L., and J. Huang designed research; L.W., J. Han, T.L., cells avoid generating crossover recombination products, which can S.D., F.X., and H.C. performed research; L.W., J. Han, and J. Huang analyzed data; and contribute significantly to genome instability (5–9). J. Huang wrote the paper. In human cells, the Bloom syndrome gene product (BLM) The authors declare no conflict of interest. helicase, inactivated in individuals with Bloom syndrome, asso- This article is a PNAS Direct Submission. ciates with TopoIIIα and RMI1/2 (BLAP75/18) to form the BTR Freely available online through the PNAS open access option. (BLM-TopoIIIα-RMI1/2) complex (10–15). This complex pro- 1L.W., J. Han, and T.L. contributed equally to this work. motes the dissolution of dHJ to yield exclusively noncrossover 2To whom correspondence should be addressed. [email protected]. – recombination products (16 18). BLM can also drive HR toward This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the formation of noncrossover products through the utilization 1073/pnas.1220921110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1220921110 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 A B C To test whether there is a direct protein–protein interaction be- tween SPIDR and BLM, we performed GST pull-down assays with Myc-SPIDR SFB-BLMNegative control SFB-SPIDRNegative controlSFB-SPIDR fi kDa SFB-BLM kDa recombinant GST-BLM fusion proteins puri ed from baculovirus- 250 Protein Number of 250 Number of Protein SFB- Morc3BLM 150 peptides peptides infected insect cells and in vitro-translated full-length SPIDR. As 150 α BLM 208 -Myc 100 100 BLM 98 F TopoIIIα 66 KIAA0146 65 -Flag shown in Fig. 1 , BLM directly interacts with SPIDR in vitro. 75 75 α RMI1 18 TopoIIIα 30 α-Flag IP: Upon the occurrence of DNA damage, BLM could form large RMI2 10 Rif1 21 50 50 Rif1 5 RMI1 15 37 37 α-Myc nuclear foci (22, 23). A physical interaction between SPIDR and FANCM 4 FIGNL1 15 KIAA0146 4 FANCM 12 Input α-Flag BLM, as demonstrated above, raises the possibility that SPIDR FANCA 4 RMI2 7 may colocalize with BLM at sites of DNA damage. Indeed, IP DEIP F Glutathione beads discrete foci of SPIDR were readily detected in cells following pulldown Input Input G IgG SPIDR SPIDR IgG BLM BLM GST + - hydroxyurea (HU) or camptothecin (CPT) treatment (Fig. 1 ). GST-BLM - + 5% IVT SPIDR Input SiRNA Con Con BLM Con Con BLM SiRNA Con Con SPIDR Con Con SPIDR Moreover, these foci colocalize with BLM foci, indicating that BLM SPIDR α-SPIDR SPIDR BLM kDa the localization of SPIDR, like that of BLM, is regulated in re- GST-BLM 250 150 G TopoIIIα TopoIIIα 100 sponse to DNA damage (Fig. 1 ). 75 RMI1 RMI1 50 To further define the binding between SPIDR and BLM, we 37 RMI2 RMI2 sought to identify the regions within SPIDR responsible for its in- GST 25 FAN1 FAN1 Coomassie staining teraction with BLM. Coimmunoprecipitation experiments revealed that SPIDR associated with BLM through its entire C terminus G SPIDR (Flag) BLM DAPI Merge Merged enlarged (residues 451–915), because deletion mutants lacking any part of this region failed to coprecipitate with BLM (Fig. S1 A and B). HU Furthermore, pull-down experiments with recombinant proteins expressed in insect cells consolidated the above notion (Fig. S1C). Conversely, using a series of overlapping BLM truncations and deletion mutants spanning its entire coding sequence, we map- CPT ped the SPIDR-binding region to residues 301–600 of BLM (Fig. S1 D–F). Fig. 1. Identification of SPIDR as a BLM-binding partner. (A and B) 293T cells SPIDR Is Required for BLM Foci Formation and Suppresses SCE. Be- stably expressing SFB-tagged BLM or SPIDR were used for TAP of protein com- cause SPIDR exists in a complex with BLM, we sought to de- plexes. Tables are summaries of proteins identified by mass spectrometry anal- termine whether SPIDR has a role in the assembly of BLM foci. ysis. Letters in bold indicate the bait proteins. (C) 293T cells were transiently Thus, three different SPIDR-specific siRNA oligonucleotides were transfected with plasmids encoding SFB-tagged BLM or Morc3 together with transfected into U2OS cells.