Breast cancer type two susceptibility protein (BRCA2) is a required component in multi-step genome maintenance processes that are coordinated in time and place. BRCA2 knock-out is lethal in mammalian cells, and a defective BRCA2 causes increased sensitivity to genotoxic agents, defective DNA repair, and reduced homologous recombination (HR) activity (Prakash et al., 2015; Sharan et al., 1997; Yu et al., 2000). One role of BRCA2 common to DNA break repair, DNA crosslink repair, and replication fork protection is delivery of RAD51 to sites where it is needed (Holloman, 2011; Sharan et al., 1997; Yuan et al., 1999). RAD51 is also an essential protein whose biochemical function is to form filaments on single-stranded DNA (ssDNA) capable of performing strand exchange reactions with homologous partners or otherwise protecting the bound DNA (Baumann and West, 1998; Heyer et al., 2010). We consider essential BRCA2 activity to involve at least (1) spatial relocation in the nucleus resulting in accumulation at sites where RAD51 is needed and (2) molecular rearrangement to release or deposit RAD51 on DNA in an active form.

Accumulation of the required proteins at the sites of DNA damage is typically defined as the appearance of foci, high local concentration of proteins, in immunofluorescence experiments. During HR, the formation of RAD51 foci is considered a critical step, and this has recently also been introduced in clinical settings as a test for HR defects in tumors (Naipal et al., 2014). The accumulation of RAD51 into foci depends on functional BRCA2 (Yuan et al., 1999). Several studies have addressed the role of different interactors and domains of BRCA2 in foci formation after DNA-damage induction (Shahid et al., 2014). The presence of Partner and Localizer of BRCA2 (PALB2) and its interaction with the N-terminus of BRCA2 are essential for the localization of BRCA2 and RAD51 to foci (Oliver et al., 2009; Xia et al., 2007; Xia et al., 2006), whereas the loss of interaction affects HR and genome stability in general (Hartford et al., 2016). In chicken DT40 cells, both N-terminal interaction with PALB2 and C-terminal DNA-binding domain (DBD) have a role in focal accumulation of BRCA2; accumulation is fully eliminated if neither domain is present (Al Abo et al., 2014). Additionally, the interaction of the BRCA2 DBD with the small DSS1 protein is required for proper localization of BRCA2 to the nucleus and for BRCA2 and RAD51 focus formation (Gudmundsdottir et al., 2004; Kojic et al., 2005; Li et al., 2006).

Accumulation of BRCA2 and RAD51 in DNA-damage-induced foci necessarily requires a change in their diffusive behavior. Single-particle tracking (SPT) in living mouse embryonic stem (mES) cells revealed that BRCA2 diffuses as multimeric complexes bound to all detectable nuclear RAD51 (Reuter et al., 2014). Individual BRCA2 particles diffuse slowly and are transiently immobile, and this immobility increases in response to DNA-damage induction (Reuter et al., 2014). Although they diffuse together, BRCA2 and RAD51 are separated at sites where they accumulate, as determined by super-resolution microscopy (Sánchez et al., 2017; Whelan et al., 2018). This suggests structural rearrangements of the complex to release RAD51. Purified BRCA2 protein shows remarkable rearrangement by RAD51 and ssDNA (Le et al., 2020; Sánchez et al., 2017; Sidhu et al., 2020). This apparent structural plasticity is a hallmark of proteins with intrinsically disordered regions (Dunker et al., 2005; Gunasekaran et al., 2003; van der Lee et al., 2014), which we hypothesize could be relevant for BRCA2 function in cells.

BRCA2 has many predicted disordered regions, which complicate understanding the functional organization and possible dynamic rearrangement of the reported structured domains. Several crystal structures of small fragments of BRCA2 are available: C-terminal BRCA2-DSS1 complex (Yang et al., 2002) (PDB ID: 1IYJ), N-terminal BRCA2-PALB2 (Oliver et al., 2009) (PDB ID: 3EU7), Brc4 BRCA2-RAD51 (Pellegrini et al., 2002) (PDB ID: 1N0W), BRCA2 phosphopeptide-Plk1 (Ehlén et al., 2020) (PDB ID: 6GY2), Brc8-2 BRCA2-RadA (Lindenburg et al., 2020) (PDB ID: 6HQU), and a BRCA2 peptide residing in exon 12 with the C-terminal armadillo-type domain of HSF2BP (Ghouil et al., 2020). Of these, the largest crystallized segment of BRCA2 is the DBD (736 amino acids), which encompasses the helical domain, tower domain, and the three oligonucleotide/oligosaccharide binding (OB) folds. There are two distinct regions of BRCA2 that bind RAD51 with different consequences (Esashi et al., 2005; Galkin et al., 2005). The eight centrally located BRC repeats bind multiple RAD51 molecules (Jensen et al., 2010). The BRC repeat-RAD51 interactions are important for localizing RAD51 into nuclear foci (Chen et al., 1999) and promoting RAD51 filament nucleation in vitro (Shahid et al., 2014). There is another RAD51-interaction domain at the C-terminus of BRCA2, which is inhibited by cell-cycle-regulated BRCA2 phosphorylation (Esashi et al., 2005). This region, the C-terminal domain (CTD) of BRCA2, which is equivalent to exon 27, has a specific role in protecting replication forks (Feng and Jasin, 2017; Lomonosov et al., 2003; Schlacher et al., 2011). The C-terminal RAD51-binding site is also suggested to stimulate RAD51-mediated recombination and stabilize RAD51 filaments (Esashi et al., 2005) while the DBD in combination with DSS1 is suggested to exchange Replication protein A (RPA) for RAD51 on ssDNA (Yang et al., 2002; Zhao et al., 2015). However, the DBD is not essential for cells to survive (Edwards et al., 2008), and although it is the most conserved part of BRCA2 (Yang et al., 2002), its exact cellular function remains elusive.

It is essential to understand how BRCA2 works to know how the regions of BRCA2, interacting with multiple partners, contribute to its function. Here we consider the influence of DBD and CTD on dynamic activities of BRCA2, DNA-damage-induced changes in diffusion and partner-binding-induced changes in protein conformation. To understand which parts of BRCA2 are responsible for dynamic localization and structural transitions, we correlated the cellular phenotypes, diffusion dynamics, and in vitro structural transitions for BRCA2 variants lacking either the DBD, the CTD, or both. We discovered that the separate domains do not play a significant role in BRCA2’s nuclear localization and diffusion dynamics or RAD51 accumulation but strongly affect protein conformational response to binding partners. The BRCA2 conformational changes correlate with cellular HR activity. Here we discuss the possible importance of BRCA2 for steps in the HR beyond its identified role in RAD51 delivery.

To determine the role of DBD and CTD in DNA-damage repair, BRCA2 mobility, and structural plasticity, we created murine cell lines and purified human BRCA2 protein lacking these domains (Figure 1A). mES lines producing tagged variants of BRCA2 (full-length; ΔDBD, containing an internal deletion of amino acids 2401–3143; ΔCTD, truncated at 3143; and ΔDBDΔCTD, truncated at 2401) were engineered by homozygous modification of the endogenous Brca2 alleles and addition of a HaloTag at the end of the coding sequence (Figure 1B, Figure 1—figure supplement 1). This allowed us to visualize BRCA2 in live or fixed cells (Los et al., 2008) and study the effect of deletions under native expression in the absence of wild-type BRCA2 (Figure 2A, Figure 3). To analyze the role of DBD and CTD in vitro, we purified variants of human BRCA2 protein containing the same deletions (Figure 1A, Figures 4 and 5).

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Full western blot images from Figure 1B.

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Original uncropped images from Figure 2A.

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Here we investigated the role of DBD and CTD on the diffusive behavior and ligand-induced structural plasticity of BRCA2. We correlated these observations with functional consequences of deleting these domains, individually and in combination, in living cells. Our panel of isogenic precision-engineered cell lines allowed us to label endogenously expressed BRCA2 directly via the HaloTag. We found that a substantial reduction in DNA-damage resistance, especially in DBD-deficient cells, was accompanied by only subtle changes in dynamics and localization of BRCA2. In contrast, the ability of purified recombinant BRCA2 to undergo structural rearrangements was strongly affected by DBD or CTD deletions (Table 1, Table 2).

Despite their adjacent location in the C-terminal part of BRCA2 (and frequent simultaneous loss due to human cancer-predisposing mutations), DBD and CTD are functionally distinct. CTD, although much shorter than the DBD, performs several distinct functions: cell-cycle-controlled phosphorylation-dependent stabilization of RAD51 filament in vitro, replication fork protection from excessive nucleolytic processing, and nuclear import. In mouse BRCA2, an additional nuclear localization signal is present at the N-terminus, but in the human protein, there is no such redundancy, which exaggerates the consequence of even short C-terminal truncations, because these produce human BRCA2 that cannot localize to the nucleus (Sarkisian et al., 2001; Spain et al., 1999). Controlled deletions, including the internal DBD deletion, allowed us to avoid some of the confounding effects complicating previously used mutant or patient cell models.

The DBD is the evolutionarily defining part of BRCA2, conserved from fungi to humans, but its function is less defined than that of other ‘younger’ BRCA2 regions. Information on DBD function focuses on its interaction with an intrinsically disordered acidic protein DSS1. Our findings reinforce the notion that despite its deep phylogenetic roots, the DBD is not what makes BRCA2 essential for general viability of animal cells. Absence of the DBD leads to significant sensitization to DNA interstrand crosslinks, PARP inhibitor, and radiation, not further exacerbated by additional CTD deletion (Figure 1). The role of DSS1 interaction remains puzzling. On the one hand, it is as conserved as the DBD itself, and it was shown to be required for BRCA2 stability and intracellular localization (Li et al., 2006); mutations in DSS1 binding phenocopy BRCA2 deficiency, as does DSS1 depletion (Zhao et al., 2015). But, on the other hand, in fungi, DSS1 is only required for DNA repair when DBD is present (Kojic et al., 2005). Similarly, in human cells, HR could be partially restored in BRCA2-deficient cells by complementation with variants lacking the DBD (Edwards et al., 2008; Siaud et al., 2011). Our results also show that cells expressing BRCA2 ΔDBD retained ~50% of HR activity (Figure 1G). In cells that lack BRCA2 DBD (ΔDBD and ΔDBDΔCTD), we do observe an increased number of RAD51 foci in the untreated condition (p<0.001), which could indicate increased replication-associated DNA damage. This, however, did not affect the growth rate of the cells. We also found that DBD contributes little to the characteristic constrained diffusion we described previously. Binding of the DBD to DNA could explain slow diffusion and frequent immobilization of BRCA2—with a higher frequency after damage induction. But the effect of loss of DBD on diffusive activity was small and comparable to loss of CTD. Therefore, we conclude that the DBD does not have a significant role in this context. In the BRCA2-containing complex, DNA-binding activity could be redundantly supplied by its interactors. For example, several BRCA2-bound RAD51 molecules provide alternative DNA interaction interfaces (Jensen et al., 2010; Reuter et al., 2014; Sánchez et al., 2017). In contrast to the DBD, the CTD is a recent vertebrate addition to BRCA2. In our assays, its deletion resulted in no phenotype (interstrand crosslink survival), intermediate phenotype (radiation and PARPi survival, HR assay, RAD51 focus number), or the same effect as DBD deletion (RAD51 focus intensity). Except for BRCA2 oligomerization (Figure 4B), deleting both domains did not result in an additive effect. It is possible that CTD deletion we created encroaches on or disturbs the structure of the DBD, and (some of) the functional consequences we attribute to the CTD deletion result from collateral damage to the DBD. The strongest argument against this is the clear separation of functions between the domains in the interstrand crosslink survival assays (Figure 1E,F) where CTD deletion has no effect. This finding also suggests that the described fork protection and RAD51 filament stabilization functions of the CTD are not essential for DNA crosslink repair. This is at odds with studies that describe the role of fork protection in crosslink repair and, in particular, with previous findings in ES cells with different CTD-disrupting Brca2 alleles (Atanassov et al., 2005; Donoho et al., 2003; Marple et al., 2006). Details of the genomic engineering strategies could account for the differences: for example, the more widely used Brca2 lex1/lex2 cells are compound heterozygotes, with a larger deletion in one of the alleles (Morimatsu et al., 1998). Another unexpected observation was that CTD deletion blocked structural rearrangements of BRCA2 upon interaction with ssDNA as efficiently as the DBD deletion. One possibility is that N-C terminal interactions that contribute to oligomerization of BRCA2 (Le et al., 2020) also affect BRCA2 structure and thereby influence the BRCA2-ssDNA interaction. Finally, it is interesting to note that BRCA2 ΔCTD cells in our hands are sensitive to IR and PARP inhibitors but are not sensitive to DNA-crosslinking agents (MMC and cisplatin); this could indicate that these structural rearrangements are more relevant in the context of BRCA2’s function in repair in two-ended double-strand breaks than during repair of DNA crosslinks, possibly due to other interacting proteins during crosslink repair.

We observed RAD51 focus formation in cell lines that were, however, deficient in HR at two-ended double-strand breaks. Despite strong sensitization to radio- and chemotherapeutic agents, only careful quantification of the numbers and intensity of RAD51 foci at multiple time points after radiation revealed subtle differences in the deletion variants. The reduced intensity of RAD51 foci in cells lacking DBD and CTD indicates that the repair process is delayed or reduced at some point beyond delivery of RAD51 by BRCA2 to the sites of damage. This suggests that RAD51 foci quantification, although useful to identify more HR-deficient samples than BRCA1/2 mutations, could occasionally return false-positive results for HR function at two-ended breaks. In and of itself, this observation is not surprising as there will be steps important for HR function downstream of RAD51 focus formation. However, in the context of employing the RAD51 focus-formation assay in pre-clinical and clinical settings with the aim to find BRCAness phenotypes, identification of additional HR markers will be of value.

The DBD and CTD of BRCA2 did markedly affect protein architecture and conformational changes in response to binding partners. These domains contributed to oligomerization, when we removed them, and in DBD and CTD deletion variants, the BRCA2 population was less oligomeric. This is in agreement with a recent study where interaction of N- and C-terminal fragments of BRCA2 is indicated to contribute to oligomerization of BRCA2 (Le et al., 2020). However, in our study, the oligomeric forms induced by RAD51 binding remain unchanged, which is likely mediated by the interaction with the intact BRC repeats. The characteristic conformational change of irregular compact particles to the extended architecture of full-length BRCA2 in response to ssDNA was severely impaired in all the investigated deletion variants. Together, the inability of the deletion variants to rearrange in vitro in the presence of ssDNA coupled with impaired HR in vivo suggests that DBD and CTD interactions of BRCA2 are important for optimal BRCA2 activity at the sites of damage. A similar regulatory function is reported for other proteins that interact with BRCA2 such as DSS1, which also affects the conformation of BRCA2 (Le et al., 2020). A recent study confirmed that regulation of oligomerization of BRCA2 is also relevant in cells, and variants of unknown significance with mutations in the DBD that reduce the binding of DSS1 show reduced nuclear localization and appear to increase/reduce oligomerization of BRCA2 (Lee et al., 2021). Altogether, these and our study suggests that regulation of RAD51 by BRCA2 is affected by conformational rearrangement of BRCA2 and is mediated at different levels by self-interaction of BRCA2 and its interaction partners.

Comparing all the molecular endpoints we analyzed (diffusion, foci, architecture), we conclude that although none correlated perfectly with the functional outcomes (survival and recombination assay), the magnitude of the effect on architectural plasticity was the closest reflection. We are only starting to tease apart the relationship between structural plasticity and cellular function. BRCA2 function may depend not so much on the existence of one structural form or another but on the lifetime of specific conformations affected by its interactors and local chromatin organization, parameters that will need to be quantified.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1–5.

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Author details

1. Maarten W Paul

   Department of Molecular Genetics, Oncode Institute, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, Netherlands

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Arshdeep Sidhu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7990-6010

2. Arshdeep Sidhu

   1. Department of Molecular Genetics, Oncode Institute, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, Netherlands
   2. Department of Radiation Oncology, Erasmus University Medical Center, Rotterdam, Netherlands

   Present address

   Arshdeep Sidhu, Division of Molecular Genetics and Cancer, Nitte University Centre for Science Education and Research, Nitte (Deemed to be University), Mangalore, India

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Maarten W Paul

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2851-1019

3. Yongxin Liang

   Department of Molecular Genetics, Oncode Institute, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, Netherlands

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

4. Sarah E van Rossum-Fikkert

   1. Department of Molecular Genetics, Oncode Institute, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, Netherlands
   2. Department of Radiation Oncology, Erasmus University Medical Center, Rotterdam, Netherlands

   Contribution

   Resources, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

5. Hanny Odijk

   Department of Molecular Genetics, Oncode Institute, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, Netherlands

   Contribution

   Resources, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

6. Alex N Zelensky

   Department of Molecular Genetics, Oncode Institute, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, Netherlands

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

7. Roland Kanaar

   Department of Molecular Genetics, Oncode Institute, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, Netherlands

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9364-8727

8. Claire Wyman

   1. Department of Molecular Genetics, Oncode Institute, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, Netherlands
   2. Department of Radiation Oncology, Erasmus University Medical Center, Rotterdam, Netherlands

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Validation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   c.wyman@erasmusmc.nl

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2549-6893

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