Recombinant Mouse Breast cancer type 2 susceptibility protein homolog (Brca2), partial

What is the functional role of BRCA2 in homologous recombination repair?

BRCA2 plays a pivotal role in the initiation of DNA repair by facilitating the loading of the repair protein RAD51 onto single-stranded DNA for homologous recombination (HR). Methodologically, this function can be studied by:

• Assessing RAD51 foci formation in response to DNA damage in cells expressing wild-type versus mutant BRCA2

• Measuring HR efficiency using reporter assays in BRCA2-deficient cells complemented with various BRCA2 constructs

• Evaluating cell sensitivity to DNA-damaging agents that specifically require HR for repair

The BRCA2-RAD51 interaction is essential for maintaining genomic stability, as cells with defective BRCA2 function show impaired HR repair capacity and subsequently accumulate genetic aberrations that can lead to tumorigenesis .

How are conditional knockout mouse models of BRCA2 generated?

Generation of conditional BRCA2 knockout models typically involves:

• Flanking the entire BRCA2 gene locus with two loxP sites alongside two halves of a selectable marker gene (such as human HPRT1 mini gene)

• Disrupting the second copy of BRCA2 by targeting a resistance gene (e.g., blasticidin) into a critical exon

• Inducing Cre recombinase expression to delete the conditional allele

• Selection of cells that have undergone successful recombination

For example, the PL2F7 mouse ES cell model was created by flanking the entire BRCA2 gene with loxP sites along with two halves of the human HPRT1 mini gene in AB2.2 ES cells lacking functional Hprt gene, making them sensitive to HAT selection. The second copy of BRCA2 was disrupted by targeting a blasticidin resistance gene into exon 11 .

This approach allows for temporal control over BRCA2 deletion, which is essential since complete BRCA2 knockout is embryonically lethal.

What experimental strategies can distinguish between pathogenic and non-pathogenic BRCA2 variants?

To classify BRCA2 variants, researchers should employ a multi-faceted approach:

• Mouse ES-cell-based functional assays that assess cellular response to DNA-damaging agents

• Homologous recombination efficiency measurements using reporter constructs

• Protein interaction studies to evaluate binding to key partners (RAD51, PALB2)

• Cell viability assays following induced DNA damage

Research has demonstrated that this approach can effectively classify variants of uncertain significance. For example, the Y42C variant, initially suspected to be pathogenic based on in vitro studies showing disrupted interaction between BRCA2's N-terminus and replication protein A (RPA), was later confirmed to be a neutral variant through ES cell-based functional assays. This highlights the importance of comprehensive functional assessment over isolated protein domain studies .

How can mouse ES-cell-based functional assays be optimized to evaluate BRCA2 mutations?

Optimizing ES-cell-based BRCA2 functional assays involves:

• Creating a conditional BRCA2 knockout ES cell line where both alleles can be inactivated

• Rescuing lethality by introducing human BRCA2-containing BAC retrofitted with a selection marker

• Introducing specific mutations into the human BRCA2 sequence

• Deleting the endogenous mouse BRCA2 using Cre recombinase

• Evaluating cellular phenotypes including:

  • Sensitivity to DNA-damaging agents (cisplatin, mitomycin C, etc.)

  • Homologous recombination efficiency

  • Chromosomal stability

  • RAD51 foci formation

This methodology has been validated through tests on known neutral variants (Y42C, D1420Y, R2784W, and K3326X) that showed no hypersensitivity to DNA-damaging agents, confirming epidemiological data suggesting these were benign polymorphisms. Conversely, the Y3308X truncation variant exhibited compromised function, consistent with its classification as a pathogenic mutation .

What methods are most effective for analyzing the BRCA1-PALB2-BRCA2 complex in mouse models?

Effective analysis of the BRCA1-PALB2-BRCA2 complex requires:

• Co-immunoprecipitation assays to verify protein-protein interactions

• Domain mapping experiments using truncated protein constructs

• Site-directed mutagenesis to identify critical residues for complex formation

• Fluorescence microscopy to examine co-localization at DNA damage sites

• Functional assays measuring HR efficiency when complex formation is disrupted

Research has revealed that PALB2 serves as a molecular scaffold between BRCA1 and BRCA2. The BRCA1-PALB2 interaction is primarily mediated via apolar bonding between their respective coiled-coil domains. Cancer-associated BRCA1 mutations can disrupt this interaction, resulting in defective homologous recombination repair. The PALB2-dependent loading of the BRCA2-RAD51 repair machinery at DNA breaks is modulated by BRCA1, forming a crucial axis for genomic stability maintenance .

How does truncation of the C-terminus of mouse BRCA2 affect protein function?

Analysis of C-terminal BRCA2 truncations should include:

• Generation of specific truncation mutations (e.g., Y3308X, E3309X, K3326X)

• Expression of truncated proteins in BRCA2-deficient cells

• Assessment of DNA repair capacity through:

  • Sensitivity to DNA crosslinking agents

  • Measurement of HR efficiency

  • Evaluation of chromosomal aberrations

  • RAD51 foci formation analysis

Research on the Y3308X variant revealed it to be hypomorphic based on the phenotype of mice with similar truncating mutations. This finding was relevant to the interpretation of the E3309X variant found in an ovarian cancer patient. The proximity of these pathogenic truncations to the known polymorphism K3326X creates analytical challenges that require careful functional assessment .

The truncation's impact depends on which functional domains are affected - C-terminal truncations may disrupt the RAD51 binding domain or nuclear localization signals, while preserving other functions.

What are the optimal methods for evaluating BRCA2 variants of uncertain significance in mouse models?

A comprehensive approach to evaluating BRCA2 variants includes:

• Selection of appropriate mouse ES cell models with conditional BRCA2 knockout capability

• Introduction of human BRCA2 variants via BAC electroporation

• Assessing cellular phenotypes through multiple assays:

  Assay Type	Methodology	Readout
  DNA Damage Sensitivity	Treatment with cisplatin, PARP inhibitors, MMC	Cell survival curve analysis
  HR Efficiency	DR-GFP reporter assay	Percentage of GFP-positive cells
  Chromosome Stability	Metaphase spread analysis	Number of chromosome breaks and radials
  Protein Interaction	Co-IP with RAD51, PALB2, BRCA1	Binding efficiency compared to wild-type

• Correlation with clinical data when available

This multi-assay approach has successfully disambiguated contradictory findings. For instance, the Y42C variant was initially predicted to be deleterious based on disrupted interaction with RPA in vitro, but functional testing in ES cells demonstrated it was benign, aligning with epidemiological evidence .

How can researchers distinguish between hypomorphic and null BRCA2 mutations in experimental models?

To differentiate between hypomorphic (partial function) and null (complete loss of function) BRCA2 mutations:

• Compare cellular phenotypes against complete BRCA2 knockout and wild-type controls

• Perform dose-response analysis with DNA-damaging agents

• Quantify the degree of HR deficiency using reporter assays

• Assess developmental phenotypes in mouse models

• Measure protein stability and cellular localization

The Y3308X truncation variant exemplifies a hypomorphic allele, as mice with similar truncations (exon 27-deletion) remain viable but display phenotypic abnormalities, while complete BRCA2 knockout is embryonically lethal. Differential sensitivity to various DNA-damaging agents can further distinguish hypomorphic from null mutations - hypomorphic mutations might show intermediate sensitivity patterns compared to wild-type and null mutants .

How can mouse BRCA2 models inform therapeutic strategies for BRCA2-deficient cancers?

Mouse BRCA2 models provide valuable platforms for therapeutic development through:

• Screening synthetic lethal interactions (e.g., PARP inhibition)

• Testing combination therapies targeting compensatory repair pathways

• Evaluating resistance mechanisms to DNA-damaging therapies

• Identifying biomarkers of response or resistance

Research methodologies should include:

• Generating tumor-bearing mice with BRCA2 deficiency

• Testing therapeutic compounds alone and in combination

• Monitoring tumor response, survival, and molecular changes

• Analyzing mechanisms of acquired resistance

Studies of BRCA2-deficient cells have already led to the development of PARP inhibitors as a therapeutic strategy. The synthetic lethality between PARP inhibition and BRCA2 deficiency provides a powerful targeted approach for tumors with compromised HR repair .

What are the methodological considerations for studying BRCA2 in the context of the BRCA1-PALB2-BRCA2 complex?

When investigating BRCA2 within the BRCA1-PALB2-BRCA2 complex, researchers should:

• Design experiments that account for the interdependence of these proteins

• Use cell models where all three proteins can be modulated individually or in combination

• Implement domain-specific mutations to dissect the contribution of each interaction

• Evaluate the impact of cancer-associated mutations on complex formation and function

PALB2 functions as the molecular adaptor between BRCA1 and BRCA2, with its N-terminus interacting with BRCA1's coiled-coil domain and its C-terminus binding to BRCA2. This architecture enables the coordinated assembly of the HR repair machinery at DNA breaks. Mutations that disrupt any component of this complex can lead to defective HR and genomic instability, even if the other components remain intact .

How should researchers interpret contradictory results between in vitro and in vivo studies of BRCA2 function?

When faced with contradictory results between in vitro and in vivo studies:

• Evaluate the biological context of each experimental system

• Consider whether isolated protein domains versus full-length protein was used

• Assess cellular compensation mechanisms present in vivo but absent in vitro

• Correlate findings with clinical and epidemiological data

• Perform additional validation using complementary approaches

The case of the Y42C variant illustrates this challenge: in vitro studies suggested it disrupted interaction with RPA, implying pathogenicity, while ES cell-based functional assays and epidemiological data indicated it was benign. This discrepancy likely arose because the in vitro study examined an isolated portion of BRCA2, whereas the ES cell assay evaluated the full-length protein in a more physiologically relevant context .

What are the methodological approaches for studying the role of mouse BRCA2 in embryonic development?

Studying BRCA2's developmental roles requires:

• Generating tissue-specific or temporally controlled knockout models

• Histological and molecular characterization of embryonic phenotypes

• Analysis of cell-cycle progression and apoptosis in developing tissues

• Evaluation of interactions with developmental signaling pathways

PALB2-deficient mouse embryos show notochord abnormalities with diffuse and discontinuous morphology, suggesting that the BRCA complex influences notochord development. Similarly, BRCA1-deficient embryos exhibit neural tube abnormalities, which are induced by notochord-derived sonic hedgehog (Shh) signaling .

These findings connect DNA repair proteins to developmental processes through mechanisms that may include:

• Protection of rapidly dividing embryonic cells from genomic instability

• Non-canonical functions in developmental signaling pathways

• Regulation of cell fate decisions during organogenesis

What methodologies can distinguish between direct and indirect effects of BRCA2 mutations on cancer susceptibility?

To differentiate direct from indirect effects of BRCA2 mutations:

• Implement conditional and tissue-specific expression of BRCA2 variants

• Perform temporal analysis of genomic instability accumulation

• Conduct transcriptomic and proteomic profiling to identify secondary pathways

• Use CRISPR-based approaches to correct or introduce specific mutations

• Develop computational models that integrate multi-omics data

Research indicates that individuals with BRCA2 mutations have up to 80% risk of developing breast cancer by age 70, but the mechanisms linking mutation to cancer development involve multiple steps . Distinguishing primary effects (direct impact on DNA repair) from secondary effects (resulting genomic instability, altered gene expression, cellular stress responses) provides insights into the long latency period between mutation acquisition and cancer manifestation.

How can high-throughput functional assays be developed to assess large numbers of BRCA2 variants simultaneously?

Development of high-throughput BRCA2 variant assessment platforms should include:

• CRISPR-based saturation mutagenesis of key BRCA2 domains

• Multiplexed reporter systems for HR efficiency

• Barcoded variant libraries coupled with next-generation sequencing

• Cell-based competitive growth assays in the presence of DNA-damaging agents

• Machine learning algorithms to predict variant pathogenicity based on functional data

Existing methods like HRM (High Resolution Melting) assays have demonstrated high sensitivity and specificity for BRCA mutation detection (100% sensitivity and >98% specificity in validation studies) , but functional characterization remains more challenging. Emerging technologies combining multiplexed variant generation with functional readouts offer promising approaches for comprehensive BRCA2 variant classification.

How should researchers integrate functional assays with clinical and epidemiological data when studying BRCA2 variants?

Integration of multiple data sources requires:

• Establishing standardized functional assay protocols with clear thresholds for pathogenicity

• Creating databases that link variant functional data with clinical outcomes

• Implementing statistical frameworks to weigh evidence from different sources

• Developing consensus guidelines for variant classification

• Forming collaborative networks to share data and methodologies

The most reliable variant classifications emerge when functional data align with clinical observations. For example, Y42C was confirmed neutral through convergent evidence from functional assays and epidemiological studies, despite contradictory in vitro findings . Similarly, functional characterization of PALB2 variants can provide insights into their clinical significance in chordoma and other cancers .