Enhancement of RAD51 recombinase activity by the tumor suppressor PALB2

Abstract

Homologous recombination mediated by RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-strand breaks induced by radiation or arising from injured DNA replication forks. The tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2: to enhance RAD51's ability to form the D loop. We show that PALB2 binds DNA and physically interacts with RAD51. Notably, although PALB2 alone stimulates D-loop formation, it has a cooperative effect with RAD51AP1, an enhancer of RAD51. This stimulation stems from the ability of PALB2 to function with RAD51 and RAD51AP1 to assemble the synaptic complex. Our results demonstrate the multifaceted role of PALB2 in chromosome damage repair. Because PALB2 mutations can cause cancer or Fanconi anemia, our findings shed light on the mechanism of tumor suppression in humans.

Figure 1. PALB2 purification

(a) Schematic summarizing the known features of PALB2 and the PALB2 species used in this work. (b) Protein purification procedures and SDS-PAGE analysis of the purified PALB2 species (1 μg each in (i) and 2 μg each in (ii)).

Figure 2. DNA binding by PALB2

(a) The DNA substrates used. The prefix denotes the identity of the oligonucleotide. (b) Full length PALB2 (5, 50, 100, 200, and 300 nM) or (c) PALB2 1-579 (5, 20, 50, 100, 200, and 300 nM) was incubated with different DNA substrates (30 nM) and then analyzed (i, ii, and iii). The results were quantified and graphed (iv). (d) and (e) PALB2 (150 nM) or PALB2 1-579 (200 nM) was incubated with radiolabeled dsDNA (30 nM) and then the nucleoprotein complex was challenged with an increasing concentration of unlabeled ssDNA, dsDNA, or D-loop (30, 60, 90, and 120 nM), as indicated. The reaction mixtures were analyzed (i), and the levels of radiolabeled nucleoprotein complex were quantified and graphed (ii). Error bars represent the standard deviation (±s.d.) calculated based on at least three independent experiments.

Figure 3. Enhancement of the RAD51-mediated D-loop reaction by PALB2 and RAD51AP1

(a) In (i), GST-PALB2 or GST (2 μg) was incubated with RAD51 (3 μg) and the PALB2-RAD51 complex was captured on glutathione resin and analyzed. In (ii), ScRad51 (3 μg) was used in place of hRAD51. In (iii), the indicated GST-tagged PALB2 fragments (4 μg) were examined for complex formation with RAD51 or ScRad51 (3 μg). S: supernatant containing unbound proteins; W: wash; E: SDS eluate of the glutathione resin. (b) Schematic of the D-loop assay is shown in (i). Panel (ii) shows D-loop reactions conducted with RAD51, PALB2, RAD51AP1, or combinations of them. ATP was omitted from the reaction in lane 11. The results were quantified and graphed in (iii). Error bars represent the standard deviation (±s.d.) calculated based on at least three independent experiments. (c) In panel (i) GST-tagged PALB2 or GST (2 μg) was incubated with MBP-RAD51AP1 (2 μg), and the PALB2-RAD51AP1 complex was captured on glutathione resin. In panel (ii), MBP-tagged RAD51AP1 (2 μg) or MBP was incubated with GST-tagged PALB2 and the PALB2-RAD51AP complex was captured on amylose resin. Elution of the protein complex from the affinity resin and subsequent analysis were conducted as in (a).

Figure 4. Effect of PALB2 on DNA damage-induced RAD51AP1 foci formation

(a) Western blots of nuclear extracts from PALB2-deficient fibroblasts (-) transduced with empty pOZC vector or with PALB2-expressing pOZC vector (+PALB2). (b) Western blots to show RAD51AP1 knockdown in the PALB2-complemented EUFA1341 cells. The signal for QM serves as a loading control. (c) Shown are representative micrographs obtained by superimposing the RAD51AP1 signal (red) of PALB2-deficient (i-ii) and PALB2-complemented (iii-vi) EUFA1341 fibroblasts onto the DAPI counter stain (blue). PALB2-deficient EUFA1341 fibroblasts are greatly impaired in RAD51AP1 foci formation after MMC treatment (i, ii), whereas PALB2-complemented EUFA1341 cells form RAD51AP1 foci (iii, iv) in response to MMC. RAD51AP1 foci can be abrogated in PALB2-complemented EUFA1341 cells by treatment with RAD51AP1 siRNA (v, vi).

Figure 5. PALB2 enhances synaptic complex assembly

(a) Shown in (i) is the schematic of the duplex capture assay. Panel (ii) shows reactions carried out with the RAD51 presynaptic filament, RAD51AP1, PALB2, or their combinations. ATP was omitted from the reaction in lane 12. The results were quantified and graphed. (b) The basis for the protection against restriction digest is explained in (i). Panel (ii) shows reactions carried out with the RAD51 presynaptic filament, RAD51AP1, PALB2, or their combinations. No restriction enzyme was added to the reaction in lane 1, and ATP was omitted from the reaction in lane 10. The results were quantified and graphed; the background of 5% (lane 2) had been subtracted from all the values. Error bars represent the standard deviation (±s.d.) calculated based on at least three independent experiments.