PALB2 is an integral component of the BRCA complex required for homologous recombination repair

Abstract

Mutations in breast cancer susceptibility gene 1 and 2 (BRCA1 and BRCA2) predispose individuals to breast and ovarian cancer development. We previously reported an in vivo interaction between BRCA1 and BRCA2. However, the biological significance of their association is thus far undefined. Here, we report that PALB2, the partner and localizer of BRCA2, binds directly to BRCA1, and serves as the molecular scaffold in the formation of the BRCA1-PALB2-BRCA2 complex. The association between BRCA1 and PALB2 is primarily mediated via apolar bonding between their respective coiled-coil domains. More importantly, BRCA1 mutations identified in cancer patients disrupted the specific interaction between BRCA1 and PALB2. Consistent with the converging functions of the BRCA proteins in DNA repair, cells harboring mutations with abrogated BRCA1-PALB2 interaction resulted in defective homologous recombination (HR) repair. We propose that, via its direct interaction with PALB2, BRCA1 fine-tunes recombinational repair partly through its modulatory role in the PALB2-dependent loading of BRCA2-RAD51 repair machinery at DNA breaks. Our findings uncover PALB2 as the molecular adaptor between the BRCA proteins, and suggest that impaired HR repair is one of the fundamental causes for genomic instability and tumorigenesis observed in patients carrying BRCA1, BRCA2, or PALB2 mutations.

Fig. 1.

Identification of PALB2 as an integral component of the BRCA protein complex. (A) Cell lysate prepared from 293T cells expressing SFB-PALB2 was subjected to TAP. PALB2-associated proteins were subsequently separated by SDS/PAGE and visualized by silver staining. Mass spectrometry analyses revealed the identities of the PALB2-associated proteins and their respective numbers of peptides obtained were shown in brackets. (B) SF9 cells were coinfected with baculoviruses expressing His-Flag-BRCA1 in addition to those expressing GST-PALB2, GST-BARD1, or GST-WTX. Cell lysates were subjected to IP by using anti-Flag (M2) beads. Immunoblotting was conducted as indicated. (C) CoIP of BRCA1 with PALB2 and BRCA2 was carried out using anti-BRCA1 antibody and immunoblotted with indicated antibodies. (D) Detection of PALB2-associated proteins; 293T cell lysates were subjected to IP using anti-PALB2 serum, and immunoblotting was carried out using antibodies as indicated. (E) Gel filtration chromatography analysis was performed using 293T cell lysate. Proteins eluted from the indicated fractions were separated by SDS/PAGE and analyzed by Western blotting using antibodies as indicated. (F) The 293T cell lysates were subjected to 3 rounds of immunodepletion using rabbit polyconal antibodies specifically raised against BRCA1, PALB2, or BRCA2. The resulting supernatants were examined for the presence of remaining BRCA1, PALB2, BRCA2, and 53BP1 content by immunoblotting using corresponding antibodies. (G) HeLa cells transfected with control or PALB2 siRNAs were subjected to IP using anti-BRCA1 antibody. Immunoblotting of whole-cell extracts or IPed samples was performed using antibodies as indicated. (H) Anti-BRCA2 IP was performed using lysates prepared from PALB2-deficient EUFA1341 cells or derivative cells reconstituted with HA-Flag-tagged PALB2. Immunoblotting of whole-cell extracts or IPed samples was performed using antibodies as indicated. (I) PALB2 bridges the BRCA1 and BRCA2 interaction in vitro. SF9 cells were infected with baculoviruses expressing His-Flag-BRCA1 and SFB-BRCA2, together with viruses expressing either GST-PALB2 or GST-PALB2 ΔN42. IP was carried out using streptavidin beads and immunoblotted with antibodies as indicated.

Fig. 2.

Determination of regions required for the BRCA1-PALB2 interaction. (A) Graphical projection of association between PALB2 (residues 9–42) and BRCA1 (residues 1393–1424) coiled-coil domains. Positions of the heptad repeat (positions a to g) were predicted by the Coil program (window = 28) (36). Boxed residues were experimentally demonstrated to be responsible for the hetero-oligomeric interaction between PALB2 and BRCA1. (B and C) PALB2 position a mutants disrupted the interactions of PALB2 with BRCA1, but not that with BRCA2. Lysates prepared from 293T cells expressing different SFB-PALB2 mutants were subjected to pull-down assay using beads coated with GST-B1F6 (B). Alternatively, PALB2-deficient EUFA1341 cells were reconstituted with SFB-tagged wild-type or mutants of PALB2, and IP was carried out using anti-Flag M2 beads (C). Immunoblotting was performed using indicated antibodies. Asterisks indicate mutants with disrupted binding with BRCA1. (D) Patient-derived BRCA1 mutations abolished the BRCA1-PALB2 association. CoIP experiments were performed using lysates derived from 293T cells expressing SFB-PALB2, together with myc-tagged wild-type or point mutants of BRCA1. Asterisks indicate mutants with disrupted binding to PALB2. (E) Interaction of wild-type or mutant BRCA1 with BACH1; 293T cell lysates expressing myc-tagged wild-type or mutant BRCA1 were subjected to IP using anti-myc antibodies, and immunoblotted using indicated antibodies.

Fig. 3.

Distinct requirements for sustained localization of BRCA1 and PALB2 at DNA damage sites. (A) IR-induced PALB2 foci formation in HCC1937 and HCC937+BRCA1 cells was analyzed by coimmunostaining using anti-Flag and anti-pH2AX antibodies. (B) Quantification of IR-induced foci formation of wild-type or mutant PALB2 in HCC1937 or HCC1937+BRCA1 cells. (C–F) Representative pictures of coimmunostaining of PALB2 (C), BRCA2 (D), and RAD51 (E) with BRCA1 in U2OS cells treated with control, BRCA1, or PALB2 specific siRNAs. (F) Quantification of the percentage of foci positive cells as described in C–E.

Fig. 4.

The BRCA1-PALB2 interaction is required for homologous recombination repair. (A) Gene conversion was assessed in U2OS DR-GFP cells treated with control, PALB2, or BRCA1-specific siRNAs. Percentage of GFP positive cells was determined by FACS analyses 48 h posttransfection. Relative gene conversion efficiency was normalized using control siRNA transfected cells, which was set as 100%. (B) Relative gene conversion was determined in BRCA1-depleted U2OS cells reconstituted with siRNA-resistant wild-type or mutant BRCA1. (C) Gene conversion was determined in PALB2-depleted cells transfected with empty vector, or reconstituted with siRNA-resistant wild-type or mutant PALB2. Relative gene conversion efficiency was normalized using cells reconstituted with wild-type PALB2, which was set as 100%. (D) A working model of the BRCA1/BRCA2/PALB2 complex in the DNA damage response.