Brca2
Created by Joanna Curran
Brca2 is a protein of 3418 amino acids and a predicted molecular weight of 390 kD. Due to its large size and fragmentation upon isolation, Brca2 was just isolated in its entirety in August 2010. The entire protein structure is too large to be modeled here, so its two major functional domains and respective ligands ( BRC4-rad51 and Brca2 C-terminus-DSS1) will be displayed. The BRC repeat segment of the Brca2 protein stretches from amino acids 990-2100, and is then followed by the 1000 amino acid long C-terminal region. The first 990 amino acids of the N-terminal region of the Brca2 protein have not been found to serve any significant biological function as of yet.
Brca2 is encoded by the 27 exon BRCA2 gene which is one of two breast cancer susceptibility genes, mutations in which cause most familial breast cancers. The Brca2 protein has been proposed to function as both a transcription regulator and in the DNA repair process by functioning in the homologous recombination pathway of DNA double-strand break repair.(5) There are no other known proteins with strong sequence similarity to Brca2, but Brca2 has eight internal BRC repeats of about 30 amino acids each, all of which are well conserved amino acid repeats between several species. Interestingly, mutations in these BRC repeats of the gene tend to be associated with higher risk of ovarian cancer than breast cancer in humans.(2)
These BRC repeats are the sites of binding between Brca2 and rad51, so their maintenance is necessary for proper function of the Brca2-rad51 complex in homologous recombination of damaged DNA. Brca2 has been observed to be localized to the nucleus, consistent with predictions that it is involved in DNA repair. It has also been observed that Brca2 interacts with rad51, a protein involved in DNA repair and recombination. Brca2 is known to mediate the binding of rad51, a recombinase enzyme, to single stranded DNA and reduce the binding of rad51 to duplex DNA, which provides evidence that Brca2 is involved in the maintenance of genomic stability. The expression of Brca2 protein has been found to be regulated by the cell cycle, as it is induced before DNA synthesis at the late G1/ early S phase.(3)
The functional importance of the Brca2 protein is its binding to rad51, a recombinase enzyme which carries out homologous recombination on damaged DNA. The protein-protein interaction takes place at the eight internal BRC repeats that are found in the Brca2 protein. It was found that the entire conserved BRC motif is needed for rad51 binding, as well as the conserved amino acids 98-339 of the rad51 protein. The interaction between Brca2 and rad51 is direct, as compared to that of brca1 and rad51 which is mediated by another protein.(9) Each of the eight BRC motifs of the human Brca2 protein has the capacity to bind one rad51 protein. The protein complex was usually found with the stoichiometry of one Brca2 protein and 4-6 bound rad51 proteins. It is only necessary to discuss the binding at one BRC motif because all rad51 Brca2 interactions are identical.
The eight BRC repeats of Brca2 are found between amino acids 990 and 2100, each repeat about 30 amino acids long. Six of the eight BRC repeats directly interact with rad51 protein, and the strongest interactions with rad51 were found to be between rad51 and BRC3 and BRC4. The crystal structure of the fourth BRC repeat (BRC4) and binding domain of rad51 is a 1.7 angstrom BRC4-rad51 complex which was studied to determine the key structural components of the binding sites. The BRC4 repeat stays in contact with rad51 from amino acids Leu-1521 to Glu-1548, a length of 28 amino acids. Within this stretch of amino acids, the Brca2 protein contains a beta-hairpin loop, beta-bulge, and an amphipathic alpha-helix. The beta-hairpin loop of the BRC segment begins at N-terminal residue Phe-1524 and ends at Val-1532, forming a loop from the 9 amino acid long stretch. This beta-hairpin loop is structured as a type I turn, and is followed by a beta-bulge at amino acid Gly-1529. This hairpin loop lies alongside of one of the beta-strands of the rad51 protein. The hairpin loop and beta-strand interface is maintained by hydrophobic interactions. Residue Phe-1524 is one point of such contact, as the aromatic ring of Phenylalanine is buried in a hydrophobic cavity formed by residues of the rad51 protein involved in the beta-strand alongside the hairpin loop. Ala-1527, also in the Brca2 hairpin loop, has hydrophobic interactions between its beta-carbon and another cavity formed by hydrophobic rad51 residues.
The BRC4-rad51 interface continues after the beta-hairpin loop and beta-bulge of the BRC4 motif. There is a short linker segment of BRC4 of 3 amino acids (Lys-1533 to Ala-1535) which wraps around a helix of the rad51 protein. Ile-1534 in the linker region interacts with hydrophobic residues of the rad51 alpha-helix. This linker region then leads into a 7 amino acid long alpha-helical segment of BRC4 from Lys-1536 to Val-1542. There are hydrophobic interactions in these two regions which maintain the interface affinity. The residues left at the C-terminus of BRC4, Lys-1543 to Glu-1548, form an irregular coil which interacts with two helices of rad51. The interface at the C-terminus is maintained by hydrophobic interactions of Leu-1545 and Phe-1546 which form a wedge that embeds itself between the two helices of rad51. There are also polar interactions that maintain the contact between rad51 and BRC4. There is a hydrogen bond between Ser-1528 of the BRC4 hairpin loop and an Asp-187 of rad51. Another hydrogen bond forms between Ser-1538 of the BRC4 alpha-helix and a Glu213 of rad51 which is a significant point of contact because it maintains the orientation of the chains for proper interaction.(6)
The BRC repeat region of Brca2 is followed by a 1000 amino acid long C-terminal region which has been found to be the most conserved portion of the Brca2 protein among species. This C-terminal region is indicated to play an important role in tumor-suppressing function as 27% of tumor-dervied missense mutations occur in this region. DSS1, a 70 amino acid long deleted in split-hand/split-foot syndrome protein, was found to bind this region of Brca2 but this interaction is still of unclear function. The structure of the Brca2-DSS1 complex was analyzed, and it was found that the Brca2 C-terminal region contains domains similar to single stranded DNA and double stranded DNA binding motifs.
Brca2 associates with single stranded DNA on duplex DNA molecules which promotes the targeting of rad51 to these single stranded DNA regions, stimulating rad51-mediated DNA strand exchange. There is evidence that Brca2 displaces a competing molecule, replication protein A (RPA), by its binding to rad51. Replication protein A also binds ssDNA, but stimulates less effective DNA repair processes than rad51. Brca2 is the key protein to homologous recombination, which when lost leaves the cells with only the means to use excision repair by RPA, a more error-prone DNA repair process.(7) The binding of Brca2 is thought to provide a rapid mechanism for the binding of rad51 to DNA, thus stimulating rad51 which forms helical nucleoprotein filaments on the DNA, catalyzing homologous DNA strand exchange to promote homologous recombination.(6)
The C-terminal segment of Brca2 contains four domains which pack linearly and successively to produce an extended structure, and a fifth domain that protrudes from the core structure of the protein. Domain one, 190 amino acids, is the helical domain as it consists of mostly alpha-helices. The helical domain contains a core structure of a four-helix cluster (alpha1, alpha8, alpha9, alpha10) and two successive beta-hairpin loops (beta1 to beta4). A 50 residue segment containing four short helices (alpha2 to alpha4) winds through the core structure. Two helices, alpha9 and alpha10, pack with another domain, OB1, through hydrophobic interactions and hydrogen bonds. The helical domain also intertwines with the N-terminus of DSS1 protein. A short 3-10 helical structure of the DSS1 protein is between hydrophobic residues of the helical domain. DSS1 then has five consecutive acidic residues which make salt bridges with the helical domain of Brca2. DSS1 follows the groove of the helical domain and then binds with the OB1 domain surface.
The next three domains are structurally homologous (OB1, OB2, OB3) and contain the oligonucleotide or oligosaccharide-binding fold (OB fold). This OB fold is also found in most other single stranded DNA binding proteins and has a canonical structure of 5- stranded beta-sheet that folds in on itself to form a beta-barrel and has a pronounced groove for ssDNA binding. The OB2, or Tower domain, has a tower-like structure because of two long, antiparallel beta-sheets that support three-helix bundle containing a helix-turn-helix motif. OB1 and OB2 pack very closely in a head-to-head arrangement through van der Waals interactions and hydrogen bonds. OB2 and OB3 pack in tandem through hydrophobic interactions and hydrogen bond contacts. All three domains pack in such a way that their individual ssDNA binding grooves create a continuous, long groove. The OB folds of the C-terminal region of Brca2 are most structurally homologous to those of Replication Protein A (RPA). RPA is a heterotrimeric protein that has two OB folds with bind ssDNA with high affinity, and another two OB folds which add to the protein's overall affinity for ssDNA. Relative to the OB domains of RPA, OB1 of Brca2 is rotated 65° and the OB domains of Brca2 pack extensively in the presence of ssDNA, whereas the RPA OB domains do not. The OB channels seen in both Brca2 and RPA are characteristic of ssDNA binding proteins. (8)
It was found that the C-terminus of Brca2 can bind ssDNA to create protein-DNA complexes. Specifically, ssDNA bound to the OB2-OB3 channel through stacking interactions between DNA bases and aromatic residues of the OB domains, hydrophobic interactions, and residue interactions with phosphate groups of DNA bases. The DNA binding of Brca2 could play a direct role in promoting rad51-mediated homologous recombination of damaged DNA. Brca2 binding to ssDNA could displace a different DNA repair protein, RPA, and facilitate the binding of rad51 to ssDNA. The binding of rad51 to the BRC repeats of Brca2, and subsequent binding of Brca2 to ssDNA, recruits rad51 to points of DNA damage and could be a way of preventing rad51 from interacting with inappropriate DNA substrates.(8)
Brca2 was found to be regulated by covalent modification through phosphorylation by CDK at Ser-3291 of the C-terminal region of Brca2. It was observed that there were low levels of phosphorylated Brca2 protein at the S and G1 phases of the cell cycle and increased levels during G2/M and during mitosis. Phosphorylation of Brca2 at Ser3291 was seen to be inversely related to Brca2 C-terminus rad51 binding ability, as phosphorylation by CDK decreased the binding affinity of Brca2 to rad51It was also found that after ionizing radiation, CDK activity decreases which leads to increased levels of non-phosphorylated Brca2. The non-phosphorylated C-terminus is now free to bind rad51 and is thought to promote the loading of rad51 to ssDNA to begin homologous recombination for DNA damage. Although the BRC repeat region and the C-terminus of Brca2 are functionally distinct, the regulation by CDK proves that the C-terminus of Brca2 may be involved in handing over rad51 to the damaged ssDNA substrate. It follows from this that the phosphorylation status of Ser3291 is very important to the interactions between the C-terminal region of Brca2 and rad51 protein, and thus crucial to the process of homologous recombination.(4)
Brca2 has also been found to function in telomere homeostasis as homologous recombination has been implicated in maintaining telomere length in cells. Homologous recombination is needed to promote efficient replication of rapidly lost telomeric sequences. Telomeres contain G-rich DNA sequences and t-loop structures, both of which are obstacles for replication forks and can cause replication forks to stall during the replication process on the telomeres. This results in fragile and disintegrated telomeres which contribute to telomeric instability. Homologous recombination by Brca2 and rad51 is thought facilitate telomere replication, therefore restoring telomere stability and integrity. This is supported by the observation that Brca2 and rad51 are associated with telomeres during the S and G2 phases of cell replication.(1)