Mutation of the breast cancer susceptibility gene, BRCA2, leads to breast and ovarian cancers. Mechanistic insight into the functions of human BRCA2 has been limited by the difficulty of isolating this large protein (3,418 amino acids). Here we report purification of full length BRCA2 and show that it both binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA). BRCA2 acts by: targeting RAD51 to ssDNA over double-stranded DNA; enabling RAD51 to displace Replication protein-A (RPA) from ssDNA; and stabilizing RAD51-ssDNA filaments by blocking ATP hydrolysis. BRCA2 does not anneal ssDNA complexed with RPA, implying it does not directly function in repair processes that involve ssDNA annealing. Our findings show that BRCA2 is a key mediator of homologous recombination, and they provide a molecular basis for understanding how this DNA repair process is disrupted by BRCA2 mutations, which lead to chromosomal instability and cancer.

SUMMARY The breast cancer susceptibility protein, BRCA2, is essential for recombinational DNA repair. BRCA2 delivers RAD51 to double-stranded DNA (dsDNA) breaks through interaction with eight conserved, ~35 amino acid motifs, the BRC repeats. Here we show that the solitary BRC4 promotes assembly of RAD51 onto single-stranded DNA (ssDNA), but not dsDNA, to stimulate DNA strand exchange. BRC4 acts by blocking ATP hydrolysis and thereby maintaining the active ATP-bound form of the RAD51-ssDNA filament. Single-molecule visualization shows that BRC4 does not disassemble RAD51-dsDNA filaments, but rather blocks nucleation of RAD51 onto dsDNA. Furthermore, this behavior is manifest by a domain of BRCA2 comprising all eight BRC repeats. These results establish that the BRC repeats modulate RAD51-DNA interaction in two opposing, but functionally reinforcing ways: targeting active RAD51 to ssDNA and prohibiting RAD51 nucleation onto dsDNA. Thus, BRCA2 recruits RAD51 to DNA breaks and, we propose, the BRC repeats regulate DNA binding selectivity.

In this work, we provide evidence of a mechanism to reinforce the strength of an icosahedral virus by using its genomic DNA as a structural element. The mechanical properties of individual empty capsids and DNA-containing virions of the minute virus of mice are investigated by using atomic force microscopy. The stiffness of the empty capsid is found to be isotropic. Remarkably, the presence of the DNA inside the virion leads to an anisotropic reinforcement of the virus stiffness by Ϸ3%, 40%, and 140% along the fivefold, threefold, and twofold symmetry axes, respectively. A finite element model of the virus indicates that this anisotropic mechanical reinforcement is due to DNA stretches bound to 60 concavities of the capsid. These results, together with evidence of biologically relevant conformational rearrangements of the capsid around pores located at the fivefold symmetry axes, suggest that the bound DNA may reinforce the overall stiffness of the viral particle without canceling the conformational changes needed for its infectivity.capsid ͉ virion ͉ nanomechanics ͉ finite element methods ͉ atomic force microscopy I nvestigation of the mechanical properties of biomolecular assemblies is important to understanding the relationship between physical structure and biological function (1) and for the application of biomaterials in the fabrication of molecular structures (2). Viruses are masterpieces of nanoengineering designed as replicating machines. In most infectious virus particles (virions), the protein shell (capsid) that encloses the nucleic acid genome reveals a minimalist architecture, based on the oligomerization of multiple copies of just one or a few types of structurally equivalent or quasiequivalent protein subunits (3, 4). However, even the most simple virion can accomplish many complex and sometimes conflicting functions during the infectious cycle (5). Virus capsids must be robust enough to protect the viral genome against physical-chemical assaults (6) but labile and͞or flexible enough to release the infectious nucleic acid into a target cell (7,8). Also, many virions accommodate a maximum amount of genetic information in the minimum space, as the nucleic acid is packed to crystal densities (9). To meet these and other stringent biological requirements, viral particles could have acquired outstanding mechanical properties, which are beginning to be revealed (10, 11). For example, it has been shown that on DNA packaging, the 29 and bacteriophage capsids can withstand internal pressures as high as 60 (12) and 20 (13) bars, respectively. Several studies have provided insights into the forces involved in DNA ejection from or packaging in phage capsids (14-16). A recent study of 29 empty capsids yielded a Young's modulus of 1.8 GPa (17), close to that of hard plastic. One of many important related aspects that have not been directly investigated yet is the influence of the enclosed genomic nucleic acid on the mechanical properties of the viral particle.The parvovirus minute virus of mice (MVM) is among ...

The human tumor suppressor protein BRCA2 plays a key role in recombinational DNA repair. BRCA2 recruits RAD51 to sites of DNA damage through interaction with eight conserved motifs of approximately 35 amino acids, the BRC repeats; however, the specific function of each repeat remains unclear. Here, we investigated the function of the individual BRC repeats by systematically analyzing their effects on RAD51 activities. Our results reveal the existence of two categories of BRC repeats that display unique functional characteristics. One group, comprising BRC1, −2, −3, and −4, binds to free RAD51 with high affinity. The second group, comprising BRC5, −6, −7, and −8, binds to free RAD51 with low affinity but binds to the RAD51-ssDNA filament with high affinity. Each member of the first group reduces the ATPase activity of RAD51, whereas none of the BRC repeats of the second group affects this activity. Thus, through different mechanisms, both types of BRC repeats bind to and stabilize the RAD51 nucleoprotein filament on ssDNA. In addition, members of the first group limit binding of RAD51 to duplex DNA, where members of the second group do not. Only the first group enhances DNA strand exchange by RAD51. Our results suggest that the two groups of BRC repeats have differentially evolved to ensure efficient formation of a nascent RAD51 filament on ssDNA by promoting its nucleation and growth, respectively. We propose that the BRC repeats cooperate in a partially redundant but reinforcing manner to ensure a high probability of RAD51 filament formation.RCA2 is a tumor suppressor protein. First identified in humans, mutations in BRCA2 cause predisposition to breast, ovarian, and other types of cancer (1, 2). It soon became apparent that BRCA2 is involved in maintaining genomic stability through its interaction with RAD51, a central component of recombinational DNA repair in humans (3). Homologous recombination serves to maintain genomic integrity in somatic cells by promoting the repair of breaks in DNA strands. BRCA2 regulates RAD51 function in DNA repair by recruiting it to the sites DNA breaks (4). For double-stranded DNA breaks, the dsDNA is resected to produce ssDNA that is rapidly bound by Replication protein A (RPA) (5-7). There, BRCA2 mediates the loading of RAD51 protein onto the RPA-ssDNA that was produced by resection (8, 9).The 3,418 amino acids (aa) of BRCA2 include several hallmark motifs that are conserved in all BRCA2-like proteins and they are regarded as critical for function. One of these key motifs is a sequence of about 35 aa in length, referred to as the BRC repeat, because it is repeated eight times in the human protein, and is separated by variable size linker regions (10-12) (Fig. S1A). These motifs are highly conserved between mammalian species (11), and they confer upon BRCA2 the ability to bind RAD51 (8). In addition to the BRC repeats, an unrelated sequence capable of binding RAD51 was mapped to the C terminus, and it controls BRCA2 function through the cell cycle (13,14). Adjacent to the BRC r...

We have analyzed the in vitro disassembly of the capsid of the minute virus of mice, and the stability of capsid chimeras carrying heterologous epitope insertions. Upon heating in a physiological buffer, empty capsids formed by 60 copies of protein VP2 underwent first a reversible conformational change with a small enthalpy change detected by fluorescence. This change was associated with, but not limited to, externalization of the VP2 N terminus. Irreversible capsid dissociation as detected by changes in fluorescence, hemagglutination activity, and electrophoretic mobility occurred at much higher temperatures. Differential scanning calorimetry in the same conditions indicated that the dissociation/denaturation transition involved a high enthalpy change and proceeded through one or more intermediates. In contrast, in the presence of 1.5 M guanidinium chloride, heat-induced disassembly fitted a two-state irreversible process. Both thermally and chemically induced dissociation/denaturation yielded a form that had lost a part of the tertiary structure, but still retained the native secondary structure. Data from chemical dissociation indicates this form may correspond to a molten globule-like monomeric state of the capsid protein. All five antigenic peptide insertions attempted in exposed loops, despite being perhaps among the least disruptive, led to defects in folding/assembly of the capsid and, in most cases, to reduced capsid stability against thermal dissociation. The results with one of the simplest viral capsids reveal a complex pathway for disassembly, and a reduction in capsid assembly and stability upon insertion of peptides, even within the most exposed capsid loops.The study of the folding, association, and disassembly of large multimeric proteins is complicated by their size, the general irreversibility of the reactions involved, and the frequent occurrence of off-pathway intermediates. However, the significant advances already made hold promise for a detailed understanding of these processes (1). Spherical virus capsids are large, multimeric proteins (2-5) and constitute attractive models for the study of the association, stability, and disassembly of very large protein complexes (for reviews see Refs. 4 and 6 -13). In addition, viral capsids are highly dynamic entities and have evolved unique structural solutions in response to the diverse, sometimes conflicting functions they must perform during the virus life cycle (4, 7, 11, 14 -16). Thus, they provide good opportunities to understand finely tuned structure-function relationships in proteins and to develop new antiviral approaches based on the inhibition of assembly or uncoating (15,17, 18).The icosahedral T ϭ 1 capsids of parvoviruses (19 -24) are formed by 60 protein subunits contributed by three nonidentical polypeptide chains that show, however, identical -fold and core sequence. VP2 is the major capsid protein and can selfassemble into empty (DNA-free) capsids (viral-like particles or VLPs).1 VP1, a minor component of natural capsids, includes...

Twenty-eight amino acid residues involved in most noncovalent interactions between trimeric protein subunits in the capsid of the parvovirus minute virus of mice were truncated individually to alanine, and the effects on capsid assembly, thermostability, and conformation were analyzed. Only seven side chains were essential for protein subunit recognition. These side chains virtually corresponded with those that either buried a large hydrophobic surface on trimer association or formed buried intertrimer hydrogen bonds or salt bridges. The seven residues are evolutionarily conserved, and they define regularly spaced spots on a thin equatorial belt surrounding each trimer. Truncation of the many side chains that were dispensable for assembly, including those participating in solvent-accessible polar interactions, did not substantially affect capsid thermostability either. However, the interfacial residues located at the base of the pores delineating the capsid five-fold axes participated in a heat-induced conformational rearrangement associated with externalization of the capsid protein N terminus, and they were needed for infectivity. Thus, at the subunit interfaces of this model virus capsid, only key residues involved in the strongest interactions are critical for assembly and stability, but additional residues fulfill other important biological roles. P rotein-protein recognition mediates many fundamental biological processes. A detailed knowledge of these processes requires the determination of the structural, energetic, and functional roles of individual amino acid residues and interactions in protein-protein interfaces. These studies have been generally undertaken by using small protein-ligand complexes or oligomeric proteins of moderate size (reviewed in ref. 1; see also refs. 2-4). In contrast, for multimeric protein complexes, such as viral capsids (5, 6) or large cellular assemblies, little is known about the specific molecular determinants of protein association and stability. Mutational studies of virus capsids, generally focused on a few specific amino acid residues, have provided important insights (7-22). However, exhaustive experimental studies on the relative importance of residues and molecular interactions in viral capsid assembly, disassembly, and͞or stability are still very limited. These studies contribute also to the understanding of protein structure-function relationships and evolution under conflictive selective constraints (22-27), and they could be exploited possibly in the design of thermostable vaccines and antiviral agents promoting capsid disassembly or interfering with assembly (23, 28-31).Many viruses, including viruses of medical or veterinary significance, have capsids of icosahedral symmetry. The icosahedral T ϭ 1 capsids of parvoviruses (32-37) are formed by 60 protein subunits that are contributed by three nonidentical polypeptide chains (VP1, VP2, and VP3). These polypeptides derive, however, from a single gene and show identical fold and core sequence (Fig. 1). In the minut...

Missense variants in the BRCA2 gene are routinely detected during clinical screening for pathogenic mutations in patients with a family history of breast and ovarian cancer. These subtle changes frequently remain of unknown clinical significance because of the lack of genetic information that may help establish a direct correlation with cancer predisposition. Therefore alternative ways of predicting the pathogenicity of these variants are urgently needed. Since BRCA2 is a protein involved in important cellular mechanisms such as DNA repair, replication and cell cycle control, functional assays have been developed that exploit these cellular activities to explore the impact of the variants on protein function. In this review we summarize assays developed and currently utilized for studying missense variants in BRCA2. We specifically depict details of each assay, including VUS analyzed, and describe a validation set of (genetically) proven pathogenic and neutral missense variants to serve as a golden standard for the validation of each assay. Guidelines are proposed to enable implementation of laboratory-based methods to assess the impact of the variant on cancer risk.

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