Plasticity of BRCA2 function in homologous recombination: genetic interactions of the PALB2 and DNA binding domains

Abstract

The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.

Figure 1. HR proficiency of mini-BRCA2 peptides.

A. Human BRCA2 domain structure. BRCA2 binds RAD51 at the eight BRC repeats in the central region of the protein and at a distinct site in the C-terminus (Cter). In addition, BRCA2 binds PALB2 at the N terminus and has a conserved DSS1 and DNA-binding domain (DBD). B. Mini-BRCA2 domain structures. Peptides contain the indicated BRCA2 domains as well as a nuclear localization signal (nls) at the N terminus and a FLAG epitope tag at the C terminus. C. DR-GFP. The DR-GFP reporter consists of two defective GFP genes . Expression of I-SceI endonuclease results in a double-strand break (DSB) at the I-SceI site in the SceGFP gene which can be repaired using the homologous sequence in the iGFP gene to generate GFP positive cells which are quantified by flow cytometry. D. BRCA2-complemented V-C8 cells have substantially higher DSB-induced HR than uncomplemented cells. P = 0.0002, two-tailed unpaired t test. A bacterial artificial chromosome (BAC) (clone RP11-777I19; BACPAC Resource Center at the Children's Hospital Oakland Research Institute in Oakland, California) which contains human BRCA2 was stably introduced into V-C8 DR-GFP cells using a linked neomycin resistance gene for selection. BRCA2 expression was verified by Western blot analysis (not shown). E. Mini-BRCA2s containing the Cter partially correct the HR defect of Brca2-deficient V-C8 hamster cells. Mini-BRCA2s that have activity relative to no mini-BRCA2 are BRC3-DBD-Cter (P<0.05), and BRC1–2-DBD-Cter and BRC1–4-DBD-Cter (P≤0.001; unpaired t test). All peptides (red asterisks) are of the expected sizes. BRC1–4-DBD-Cter corrects the HR defect as well or better than BRC1–2-DBD-Cter despite being poorly detected. F. Comparison of HR activity at different levels of BRCA2 peptide expression. The BRC1–2-DBD-Cter expression vector was transfected at the usual amount (50 µg) or at a reduced amount (10 µg). HR levels were similar in both cases (P = 0.82). G. Mini-BRCA2s with multiple BRC repeats are more active in HR than those with single BRC repeats. Relative to BRC1–2-DBD-Cter, P≤0.0001 for single repeat mini-BRCA2s, except for BRC2-DBD-Cter where P = 0.003. All mini-BRCA2s have significant activity relative to no mini-BRCA2 (P≤0.005), except BRCΔ3-DBD-Cter (P = 0.248).

Figure 2. Mini-BRCA2 activity requires an intact DBD.

A. BRCA2-ssDNA interface in the DBD. BRCA2 OB2 (red) and OB3 (teal) folds interact with oligo(dT) (green) . i) A key interaction in OB2 is at a tryptophan residue (gold) (W2990 human/W2909 mouse) which stacks with the first two oligo(dT) bases, in parallel with the first base and at an angle with the second. ii) A lysine (gold) (K2971 human/K2891 mouse), also in OB2, interacts with the edges of the second and third bases. For the relevant interacting nucleotides and lysine, the nitrogens and oxygens are blue and red, respectively. Dotted white lines indicate the interaction. Detail of the mouse BRCA2 DBD^ΔTower-DSS1-oligo(dT) ternary complex extracted from the RCSB Protein Data Bank, accession code 1MJE. B. Mutations of ssDNA binding residues in mini-BRCA2 impair HR. The mini-BRCA2 in these assays is BRC1–2-DBD-Cter. For values relative to wild-type mini-BRCA2, P≤0.0001; for values relative to no mini-BRCA2, P≤0.002; for W2990A compared with K2971A, P = 0.0061. Western blots of the transiently expressed mini-BRCA2s are shown beneath the graph. C. BRCA2 DBD crystal structure showing the tower domain arising from OB2, with a three-helix bundle (3HB) at the apex of the tower which is proposed to bind dsDNA . Deletions which remove a large part of the tower (Tdel) or only the 3HB (Adel) are indicated on the structure. The structure of the DBD was solved in a co-crystal with the 70 amino acid protein DSS1 (orange). DSS1 binds in an extended conformation to the helical domain (HD, magenta) and OB1 (green), at the opposite side of the structure from oligo(dT) (not shown). Mouse BRCA2 DBD-DSS1 structure extracted from the RCSB Protein Data Bank, accession code 1MIU. D. Domain structures of mini-BRCA2s modified within the tower. For Tdel, the seven amino acid linker provides flexibility for the first tower helix and the disordered region to fold back and reconnect with OB2. For Adel, the three amino acid linker at the site of the deletion is predicted to connect the ends of the tower without distorting the structure. Boundaries of BRCA2 amino acids deleted are indicated. E. Deletion of the tower domain or just the 3HB in mini-BRCA2 abrogates HR. For values relative to wild-type mini-BRCA2, P≤0.001; relative to no mini-BRCA2, neither Tdel nor Adel is statistically significant (P = 0.53 and 0.58, respectively).

Figure 3. Mutation of a DSS1-interacting residue or the Cter RAD51-interacting residue S3291 significantly reduces the HR activity of mini-BRCA2.

A. Detail of the BRCA2-DSS1 interface. BRCA2 and DSS1 interact through both charged and hydrophobic residues . For the former, basic residues in BRCA2 interact with acidic residues in DSS1 (e.g., K2630 human/K2551 mouse interacts with DSS1 D17; white). Detail of the mouse BRCA2 DBD-DSS1 structure extracted from the RCSB Protein Data Bank, accession code 1MIU. B. Mutation of BRCA2 K2630 in mini-BRCA2 reduces DSS1 interaction. Immunoprecipitation of myc-tagged DSS1 brings down FLAG-tagged wild-type mini-BRCA2 but K2630A or K2630D-mutated mini-BRCA2 to a much lower or undetectable level, respectively. C. Mutation of a DSS1 interacting residue in mini-BRCA2 abrogates HR. For values relative to wild-type mini-BRCA2, P≤0.001; relative to no mini-BRCA2, neither K2630A nor K2630D is statistically significant. D. Mutation of the Cter RAD51 binding site in mini-BRCA2 impairs HR. For values relative to wild-type mini-BRCA2, P≤0.0001; relative to no mini-BRCA2, P≤0.0001.

Figure 4. Midi-BRCA2s containing an intact PALB2 binding site restore HR levels.

A. Midi-BRCA2 domain structures. Midi-BRCA2s contain the PALB2-interacting domain at the N terminus. Midi-BRCA2-2 differs from full-length BRCA2 primarily in being deleted for the last 6 BRC repeats; it also contains a small deletion N-terminal to BRC1. B. Western blot analysis showing expression of the transiently expressed mini and midi-BRCA2s. C. Midi-BRCA2s are more proficient at HR than mini-BRCA2. Relative to mini-BRCA2, P≤0.0001 for midi-BRCA2s. D. BRCA2 W31 is a key PALB2 binding residue. Mutation of midi-BRCA2 W31 disrupts binding to PALB2. Immunoprecipitation of FLAG-tagged wild-type or I27C midi-BRCA2 efficiently brings down PALB2 while FLAG-tagged W31C midi-BRCA2 precipitation of PALB2 is significantly impaired. BRCA2 W31 extends from a short helix into a hydrophobic pocket of the PALB2 ß-propeller; the indole nitrogen (light blue) also supports a polar interaction with a water bridge to PALB2 S1065 in the PALB2 pocket (dotted line) (accession code 3EU7). Only a portion of the PALB2 ß-propeller is shown. E. Mutation of the PALB2 binding site (W31C) in midi-BRCA2-1 reduces but does not abolish HR activity. For values relative to wild-type midi-BRCA2-1, P≤0.0001 except for midi-BRCA2 I27C, which is not significant; for mini-BRCA2 relative to midi-BRCA2-1 W31C, P = 0.13. Western blot analysis shows expression of the transiently expressed mini and midi-BRCA2s. F. Mutation of the PALB2 binding site (W31C) in midi-BRCA2-2 reduces but does not abolish HR activity. Relative to wild-type midi-BRCA2-2, P≤0.005 for the W31C and/or S2391A mutants; for midi-BRCA2-2 W31C relative to no midi-BRCA2-2, P≤0.0001 whereas W31C+S3291A is not significantly different from no midi-BRCA2-2 (P = 0.13). Western blot analysis shows expression of the transiently expressed peptides.

Figure 5. Interaction with PALB2 partially compensates for mutations in the DNA binding domain.

A. Midi-BRCA2s with mutations in ssDNA contact residues retain substantial HR activity. P≤0.0001 compared with no BRCA2 peptide. i) Compared with wild-type midi-BRCA2-1, P values are 0.002 (W2990A), 0.05 (K2971A), and 0.01 (K2971E). ii) Compared with wild-type midi-BRCA2-2, P values are 0.0004 (W2990A), 0.15 (K2971A), and 0.0002 (K2971E). Western blot analysis shows expression of the transiently expressed peptides. B. Midi-BRCA2s with deletion of the tower domain or just the three-helix bundle retain partial HR activity. P≤0.0002 compared with no BRCA2 peptide. Compared with the respective wild-type proteins, P values for mini-BRCA2 mutations are 0.003 (Tdel) and 0.0008 (Adel) (Figure 2E), for midi-BRCA2-1 are 0.002 (Tdel) and 0.02 (Adel), and for midi-BRCA2-2 are 0.0001 (Tdel, Adel). Western blot analysis shows expression of the transiently expressed peptides.

Figure 6. Interaction with PALB2 partially compensates for point mutations in the Cter that interfere with RAD51 binding, but cannot compensate for mutations that interfere with DSS1 binding.

A. Mutation of a DSS1 interacting residue in midi-BRCA2 abrogates HR. For midi-BRCA2-2, K2630A and K2630D mutations in comparison with no BRCA2 peptide are not significantly different (P = 0.08 and 0.52, respectively), while compared with the respective wild-type proteins, they are (mini-BRCA2, P≤0.001, as in Figure 2H; midi-BRCA2-2, P<0.0001). B. Midi-BRCA2s with mutation of the Cter RAD51 binding site retain partial HR activity. P≤0.0001 compared with no BRCA2 peptide or with the respective wild-type proteins. Western blot analysis shows expression of the transiently expressed peptides.

Figure 7. BRCA2 DBD is not required for HR in peptides that bind PALB2.

A. Tr-BRCA2 domain structures. All Tr-BRCA2 peptides are deleted for C-terminal BRC repeats and the BRCA2 DBD at the indicated residues. TrBRC3 and TrBRC5 are additionally deleted for the Cter. TrBRC5-Cter corresponds to a BRCA2 peptide expressed in a PARP inhibitor-resistant revertant of Capan-1 cells (PIR2; [40]). B. TrBRC-Cter peptides are functional for HR whereas TrBRC peptides are not. The RAD51 binding site at S3291 contributes to HR activity but is not the sole determinant. P≤0.0001 for TrBRC-Cter compared with the respective no Cter or S3291-mutated Cter. C. Disruption of PALB2-binding by W31C mutation (right) is associated with impaired TrBRC5-Cter HR activity (left). P = ≤0.0001 for TrBRC5-Cter compared with the W31C mutation.

Figure 8. BRCA2 peptides functional in HR.

A. Micro-BRCA2 domain structure. Micro-BRCA2 contains three domains – the PALB2 interaction site, BRC1–2, and the Cter, whereas zippo-BRCA2 is deleted for the BRC repeats. B. Western blot analysis showing expression of the indicated BRCA2 peptides. C. Comparison of HR activity of various BRCA2 peptides. Micro-BRCA2 is more active than mini-BRCA2 (P = 0.0024), whereas zippo-BRCA2 is not active. D. Summary of BRCA2 peptides and their relative HR activity. E. Schematic of peptides active in HR. While full-length BRCA2 has optimal HR activity, BRCA2 peptides can be derived which have substantial activity. Those that bind PALB2, such as micro-BRCA2, are active even with a deleted DNA binding domain (DBD). In the absence of PALB2 binding, an intact DBD is critical for HR activity. BRC fusion to RPA, however, can bypass the requirement for the DBD.