Mapping the physical and functional interactions between the tumor suppressors p53 and BRCA2 - PubMed (original) (raw)

Mapping the physical and functional interactions between the tumor suppressors p53 and BRCA2

Sridharan Rajagopalan et al. Proc Natl Acad Sci U S A. 2010.

Abstract

p53 maintains genome integrity either by regulating the transcription of genes involved in cell cycle, apoptosis, and DNA repair or by interacting with partner proteins. Here we provide evidence for a direct physical interaction between the tumor suppressors p53 and BRCA2. We found that the transactivation domain of p53 made specific interactions with the C-terminal oligonucleotide/oligosaccharide-binding-fold domains of BRCA2 (BRCA2(CTD)). A second distinct site situated on the p53 DNA-binding domain, bound to a region containing BRC repeats of BRCA2 (BRCA2([BRC1-8])) and may contribute synergistically for high affinity association of intact full-length proteins. Overexpression of BRCA2 and BRCA2(CTD) suppressed the transcriptional activity of p53 with a concomitant reduction in the expression of p53-target genes such as Bax and p21. Consequently, p53-mediated apoptosis was significantly attenuated by BRCA2. The observed physical association of p53 and BRCA2 may have important functional implications in the p53 transactivation-independent suppression of homologous recombination and suggests a possible interregulatory role for both proteins in apoptosis and DNA repair.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

(A) Domain organization of p53 and BRCA2. The functional domains of p53 comprise an intrinsically unstructured transactivation domain TAD (1–93), specific DNA-binding core domain DBD (94–292), oligomerization domain (325–355), and C-terminal negative regulatory domain (362–293) (28). BRCA2 is a 3,418 amino acid protein consisting of an N-terminal transactivation domain (23–105), eight central BRC repeats of ∼30 amino acids each (987–2113), and a conserved C-terminal domain BRCA2CTD (2479–3152). The latter comprises an α-helical domain and three OB domains. (B) SDS-PAGE showing the purity of different BRCA2 domains used in pull-down experiments. (C) Interaction of p53 and BRCA2. Nickel pull-down of his-tag purified BRCA2CTD domains with flp53. Immunoblotting by using PAb1801 showed that OB2 + OB3, OB2, and OB3 interact with p53. *D) Nickel pull-down of truncated domains of p53 with his-tagged OB2 + OB3 construct of BRCA2. Immunoblots using PAb2433 (Abcam, specific for 277–296 aa of p53) and PAb1801 (Abcam, specific for 46–55 aa of p53) identified that OB2 + OB3 interacts with the transactivation domain of p53 (p53 TAD, 1–93).

Fig. 2.

Binding of OB2, OB3, and OB2 + OB3 to p53 TAD. (A) Alexa fluor 546-labeled p53 TAD was titrated with OB2 + OB3, OB2, and OB3 in separate experiments. The data were fitted to a simple one-state binding model. (B) Binding isotherm of full-length p53 with OB3 as measured by using ITC in a buffer containing 25 mM Hepes, 150 mM NaCl, 3 mM DTT, pH 7.4 at 293 K. (C) Overlay of 1H15N HSQC of labeled p53 TAD (1–93) in the presence (Blue) and absence (Red) of OB3.

Fig. 3.

Binding of p53 TAD2 to BRCA2CTD. (A) Competition titration of a complex of coumarin-labeled p53 TAD2 (35–57)-OB3 with unlabeled ssDNA. (B) Homology model of human BRCA2 OB2 and OB3 domains with DNA placed in the same position as in the structure of the mouse BRCA2. Residues that can make a contact with DNA and have been mutated in this study are shown as sticks. (C) Superimposition of modeled human BRCA2-OB2 bound to ssDNA with RPA bound to p53 TAD. The color scheme is as follows: BRCA2-OB2 shown in green, RPA in blue, DNA in yellow, and p53 TAD in pink. DNA is placed in the same position as in the structure of the mouse BRCA2.

Fig. 4.

Interaction of BRCA2[BRC1-8] with the DNA-binding domain of p53. (A) Nickel pull-down experiment of his6 BRCA2[BRC1-8] with different domains of p53. Immunoblots were developed by using ab2957 (Abcam, specific for 1324–1347 of human BRCA2). (B) Overlay of 1H15N HSQC of labeled p53 DNA-binding domain (94–312) in the presence (Blue) and absence (Red) of BRCA2[BRC1-8]. (C) Chemical shift mapping of the residues on the p53 DNA-binding domain (PDB ID code 2AC0) that are perturbed upon BRCA2[BRC1-8] binding. p53 DBD is shown in green and DNA in black. Residues that showed chemical shift changes and those that disappeared are shown in red and blue, respectively. Selected residues that showed significant chemical shift perturbations are highlighted in magenta.

Fig. 5.

Transcriptional and apoptosis assays. (A) H1299 cells with inducible p53 expression were transfected with BRCA2, BRCA2CTD, BRCA2 ΔCTD, or pcDNA (+)(empty) vectors as indicated. Expression of p53 was induced by placing the cells in medium containing no tetracycline. Bars 1–4 represent experiments where p53 was not induced (∗P < 0.05). (B) Caspase-3 activity assay monitoring the apoptosis in H1299 cells transfected with BRCA2, BRCA2CTD, BRCA2 ΔCTD, and pcDNA (+)vectors as indicated in A (∗P < 0.05). (C) Expression levels of p53 target genes, p21 and Bax, when transfected with empty vector, BRCA2CTD, BRCA2, and BRCA2 ΔCTD. (D) Western blot showing the overexpression of BRCA2 when tranfected in H1299 cells. p53 levels remained unaffected by BRCA2 overexpression as indicated by Western blot. The numbers represent the cell extracts from the table in A.

Fig. 6.

Role of p53 in HR repression. p53 can suppress the early stage of HR by physical association with HR proteins such as RPA, BRCA2, and RAD51. Both p53-mediated cell-cycle arrest (p53-transactivation-dependent) and repression of HR (p53-transactivation-independent) may prevent cell proliferation under circumstances where DNA damage is left unrepaired.

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Mapping the physical and functional interactions between the tumor suppressors p53 and BRCA2 - PubMed (original) (raw)