Breast cancer-associated missense mutants of the PALB2 WD40 domain, which directly binds RAD51C, RAD51 and BRCA2, disrupt DNA repair - PubMed (original) (raw)

Breast cancer-associated missense mutants of the PALB2 WD40 domain, which directly binds RAD51C, RAD51 and BRCA2, disrupt DNA repair

J-Y Park et al. Oncogene. 2014.

Abstract

Heterozygous carriers of germ-line mutations in the BRCA2/FANCD1, PALB2/FANCN and RAD51C/FANCO DNA repair genes have an increased lifetime risk of developing breast, ovarian and other cancers; bi-allelic mutations in these genes clinically manifest as Fanconi anemia (FA). Here, we demonstrate that RAD51C is part of a novel protein complex that contains PALB2 and BRCA2. Further, the PALB2 WD40 domain can directly and independently bind RAD51C and BRCA2. To understand the role of these homologous recombination (HR) proteins in DNA repair, we functionally characterize effects of missense mutants of the PALB2 WD40 domain that have been reported in breast cancer patients. In contrast to large truncations of PALB2, which display a complete loss of interaction, the L939W, T1030I and L1143P missense mutants/variants of the PALB2 WD40 domain are associated with altered patterns of direct binding to the RAD51C, RAD51 and BRCA2 HR proteins in biochemical assays. Further, the T1030I missense mutant is unstable, whereas the L939W and L1143P proteins are stable but partially disrupt the PALB2-RAD51C-BRCA2 complex in cells. Functionally, the L939W and L1143P mutants display a decreased capacity for DNA double-strand break-induced HR and an increased cellular sensitivity to ionizing radiation. As further evidence for the functional importance of the HR complex, RAD51C mutants that are associated with cancer susceptibility and FA also display decreased complex formation with PALB2. Together, our results suggest that three different cancer susceptibility and FA proteins function in a DNA repair pathway based upon the PALB2 WD40 domain binding to RAD51C and BRCA2.

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Conflict of interest statement

CONFLICT OF INTEREST

MF and HH may receive royalties based on a licensing agreement with Myriad Genetics, Inc. for the use of RAD51C as a cancer susceptibility gene. All other authors declare that they have no potential conflicts of interest.

Figures

Figure 1

Figure 1. RAD51C interacts with PALB2 and BRCA2

(A) Silver stained gel indicating components of a RAD51C complex identified by mass spectrometry following immunopurification from HeLa S3 cells. RAD51C was expressed with a N-terminal His6-Flag (HF) epitope tag. The gel was cut into four sections as indicated (left) and mass spectrometry was performed on each piece. The number of unique peptides identified, following subtraction of common peptides isolated from a mock-purified control, is shown in parentheses. (B) Immunoblot demonstrating that PALB2 and BRCA2 co-immunoprecipitate with HF-RAD51C. (C) Immunoblot demonstrating that RAD51C and XRCC3 co-immunoprecipitate with HF-PALB2. (B–C) Immunoprecipitates were prepared using M2 anti-Flag beads and extracts from HeLa (B) and EUFA1341 cells complemented with HF-PALB2 (C). (D) PALB2, BRCA2, RAD51C and RAD51 form a protein complex, as determined by depletion of a complex that could be immunoprecipitated with HF-RAD51C after prior immunodepletion of RAD51. HeLa cells were first incubated with anti-RAD51 or normal rabbit (Cont.) antisera. Supernatants were then incubated with anti-M2 agarose to immunoprecipitate HF-RAD51C and were immunoblotted with the indicated antibodies. (B–D) Immunoprecipitates represent 200-fold the levels loaded for Input lanes. The positions of HF-RAD51C and endogenous RAD51C, or RAD51, are indicated by arrowheads.

Figure 2

Figure 2. RAD51C directly binds to the WD40 domain of PALB2

(A) Schematic diagram of PALB2 and selected mutants. Hatched boxes indicate WD40 repeats of PALB2. (B) Wild-type PALB2 and the Y551X truncation mutant containing N-terminal Flag and HA epitope tags were transiently transfected into 293T cells. Immunoprecipitates represent 500-fold the levels loaded for Input lanes. (C) Cell lysates were prepared from EUFA1341 cells reconstituted with wild-type PALB2 or the ΔC mutant truncated after P1097. The position of RAD51C is indicated by an arrowhead. (B–C) Cell lysates were immmunoprecipitated with α-Flag beads, followed by immunoblotting with the indicated antibodies. (D) Analysis of direct binding using bacterially-expressed maltose-binding protein (MBP) alone or MBP fused to the WD40 domain of PALB2 (amino acids 859–1186). Bacterially-expressed His6 alone, or fused to RAD51C, was incubated with MBP or MBP-PALB2 immobilized on maltose beads. Bound RAD51C was detected by immunoblotting with anti-His antibodies following elution.

Figure 3

Figure 3. Analysis of the positions and potential effects of breast cancer mutations in the PALB2 WD40 domain

(A) A ribbon diagram of the WD40 domain of PALB2 shown in grey (PDB ID 3EU7). The blades are numbered 1 to 7 and colored light grey to dark grey. The missense mutations/variants (L939, T1030 and L1143) found in breast cancer patients are shown in stick representation and in pink. (B–C) Surface representation of the region of PALB2 centered on Leu939 and Leu1143, respectively, colored in pink showing how both hydrophobic residues are solvent exposed. (D) Details of residues surrounding T1030 of blade 4. Residues E1011 and M1032, which make hydrogen bonds with the hydroxyl group of T1030, are highlighted. The figure was prepared with Pymol (

http://www.pymol.org/

).

Figure 4

Figure 4. Different patterns of in vitro binding of RAD51C, BRCA2, RAD51, and XRCC3 to disease-associated missense mutants of PALB2 suggest that the PALB2 WD40 domain may scaffold these proteins into a complex

(A) A representative in vitro binding experiment. MBP alone, or MBP-fused to the wild-type PALB2 WD40 domain or to different breast cancer-associated mutants/variants of this domain, were expressed in bacteria, purified, and immobilized on maltose beads. Isolated MBP-WD40 proteins or MBP alone are shown as “Inputs”. A GST-tagged BRCA2 fragment (amino acids 1–75), or His-tagged RAD51C, RAD51, or XRCC3, were purified from bacteria and incubated with the purified MBP fusion proteins. Proteins present in the MBP fusion protein pull-down were detected with anti-GST or Anti-His antibodies, as appropriate. GST alone served as a negative control for GST-BRCA2, while His alone served as a negative control for His-fusion to RAD51C, RAD51, or XRCC3. (B) A graph showing quantification of binding from three independent experiments. Values for each HR protein were determined by densitometry and adjusted relative to the levels of each form of PALB2 in the Input lanes and are shown normalized to the amounts that immunoprecipitated with the wild-type PALB2 WD40 domain. The mean and standard deviation are shown for each value (* = P<0.05; ** = P<0.01).

Figure 5

Figure 5. The breast cancer-associated T1030I mutant of PALB2 is unstable

(A) Wild-type PALB2 or the L939W, T1030I, and L1143P mutants/variants were retrovirally expressed in EUFA1341 cells along with a N-terminal Flag-HA epitope tag. The level of each protein was determined by immunoblotting with α-HA antibodies. Actin was used as a loading control. (B) To examine relative rates of turnover, new protein synthesis was inhibited in EUFA1341 cells reconstituted with Flag-HA tagged wild-type PALB2 or with the T1030I mutant by treating with 40 μg/ml cycloheximide. Cell lysates were prepared at the indicated time points. (C) EUFA1341 cells reconstituted with wild-type PALB2 or the T1030I mutant were treated with 10 μM MG132 and harvested at the indicated time points. (D–E) Quantification of the levels of wild-type PALB2 or the T1030I mutant following treatment with cycloheximide (D) or MG132 (E) at various time points was determined by densitometry of immunoblots. Results for each protein were normalized to the values at 0 hr (D) or 2 hr (E) of treatment. (F) Levels of mRNA of wild-type or mutant PALB2 in reconstituted EUFA1341 cells were analyzed by RT-PCR. Primers that detected Flag-HA-tagged PALB2 but not endogenous PALB2 were utilized. GAPDH was used as a control for the levels of an unrelated endogenous protein.

Figure 6

Figure 6. The L939W and L1143P mutants of the PALB2 WD40 domain are associated with defective DSB-initiated HR and with increased sensitivity to ionizing radiation

(A) Gene conversion assays were performed in U2OS-DR cells stably expressing wild-type PALB2, or the L939W or L1143P mutants, and depleted of endogenous PALB2 using a siRNA directed against its 3′-UTR. GFP-positive cells were quantified by flow cytometry. Each value represents the average of three independent experiments performed in duplicate. The data shown are the mean and standard deviation of three independent replicates (* = P < 0.05; ** = P < 01). (B) The percentage of cells with RAD51 foci at 16 hr after exposure to 10 Gy IR. U2OS-DR cells were stably reconstituted with the different forms of PALB2, as indicated, and endogenous PALB2 was depleted as described above. Each value represents the mean and standard deviation of 3 counts of 150 or more cells each with 3 or more RAD51 foci (* = P < 0.05). (C) IR colony assay performed in EUFA1341 cells reconstituted with wild-type PALB2, the L939W or L1143P mutants, or with vector alone. Cells were treated with the indicated doses of IR. Differences for EUFA1341 cells reconstituted with the vector or with either mutant, as compared to cells corrected with WT PALB2, were statistically significant (P < 0.05) at doses of IR ranging from 1–8 Gy. (D–E) Wild-type PALB2, the L939W or L1143P mutants, or the vector alone, were stably expressed in EUFA1341 cells along with a N-terminal Flag-HA epitope tag. Levels of associated HR proteins in extracts (Input), or in immunoprecipitates (IP) prepared using α-Flag M2 agarose beads, are shown in (D). These results are representative of two independent experiments. The position of RAD51C is indicated by an arrowhead. Immunoprecipitates represent 200-fold the levels loaded for Input lanes. Quantification of the levels of BRCA2, RAD51C or RAD51 which immunoprecipitated with the WT, L939W or L1143P forms of PALB2 in two independent experiments (E). Levels of immunoprecipitating protein were normalized to the input for each form of PALB2 and each interacting protein. The average +/− S.D. are shown relative to the values for PALB2-WT (which was set to 100%). Statistical significance: * = P < 0.05; ** = P < 0.01.

Figure 7

Figure 7. Disease-associated mutants of RAD51C have decreased interactions with BRCA2 and PALB2

(A) Wild-type RAD51C, the L138F and D159N mutants found in breast cancer patients, or the R258H mutant found in a Fanconi anemia patient, were transiently expressed in 293T cells along with a N-terminal Flag-HA epitope tag. The levels of protein in extracts (Input), or in immunoprecipitates prepared using α-Flag M2 agarose beads (IP), were determined by immunoblotting with the indicated antibodies. Immunoprecipitates represent 500-fold the levels loaded for Input lanes.

Comment in

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