BRIP1 Recombinant Monoclonal Antibody

What is BRIP1 and what are its alternative names in scientific literature?

BRIP1 (BRCA1-interacting protein 1) is known by several alternative names in scientific literature, including FANCJ, BACH1 (BRCA1-associated C-terminal helicase 1), Fanconi anemia group J protein, DNA 5'-3' helicase FANCJ, and BRCA1-interacting protein C-terminal helicase 1 . These nomenclature variations reflect its discovery context and functional roles in different cellular pathways. When designing experiments or searching literature, researchers should consider all these designations to ensure comprehensive coverage of relevant information.

What is the molecular structure and cellular localization of BRIP1?

BRIP1 is a 1249 amino acid protein with a molecular mass of approximately 140.9 kDa . It contains DNA helicase domains characteristic of the DEAH helicase family, particularly at its N-terminus, which share substantial sequence homology to the catalytic and nucleotide-binding domains of other DEAH family members . Subcellularly, BRIP1 primarily localizes to the nucleus where it participates in DNA repair processes, but it can also be found in the cytoplasm . The protein undergoes alternative splicing, generating at least two different isoforms . This information is crucial for selecting appropriate antibodies that target conserved regions across isoforms.

What are the key biological functions of BRIP1 in cellular processes?

BRIP1 functions as a DNA-dependent ATPase and 5'-3' DNA helicase required for the maintenance of chromosomal stability . It plays critical roles in multiple DNA repair pathways, including: (1) repair of DNA double-strand breaks through homologous recombination in a manner dependent on its association with BRCA1 ; (2) participation in the Fanconi anemia pathway downstream of FANCD2 ubiquitination ; and (3) repair of abasic sites at replication forks by promoting the degradation of DNA-protein cross-links . Additionally, BRIP1 can unwind RNA:DNA substrates and G-quadruplex DNA structures, with the latter unwinding requiring a 5'-single stranded tail . These diverse functions highlight its importance in genome integrity maintenance.

How does BRIP1 contribute to DNA double-strand break repair?

BRIP1 contributes to DNA double-strand break repair primarily through its association with BRCA1, which is essential for homologous recombination (HR) . This interaction depends on the cell cycle-regulated phosphorylation of BRIP1 at Serine 990 . Once activated, the BRIP1-BRCA1 complex is required for timely repair of DNA double-strand breaks and for DNA damage-induced checkpoint control during the G2/M phase of the cell cycle . BRIP1's helicase activity facilitates DNA unwinding at break sites, which is necessary for the subsequent steps of homologous recombination. When designing experiments to study this process, researchers should consider cell synchronization techniques to capture the cell cycle-dependent aspects of BRIP1 function.

What is the role of BRIP1 in the Fanconi anemia pathway?

As FANCJ, BRIP1 acts late in the Fanconi anemia (FA) pathway, functioning downstream of FANCD2 ubiquitination . In this pathway, BRIP1 helps resolve complex DNA lesions, particularly interstrand crosslinks that block replication. Mutations in the BRIP1 gene cause Fanconi anemia complementation group J, highlighting its essential role in this DNA repair pathway. Researchers investigating BRIP1's function in the FA pathway should consider experimental designs that introduce DNA crosslinking agents such as mitomycin C or cisplatin, followed by analysis of BRIP1 recruitment to damage sites and interaction with other FA proteins.

How does BRIP1's helicase activity contribute to genomic stability?

BRIP1's 5'-3' DNA helicase activity is fundamental to maintaining genomic stability through several mechanisms . It unwinds various DNA structures, including G-quadruplexes, which can form at telomeres and other G-rich regions . By resolving these potentially problematic DNA secondary structures, BRIP1 prevents replication fork stalling and collapse. Additionally, BRIP1 catalyzes the unfolding of DNA-protein cross-links, specifically involving HMCES, which exposes the underlying DNA and enables cleavage of the DNA-protein adduct by the SPRTN metalloprotease . These activities collectively prevent the accumulation of DNA damage that could otherwise lead to chromosomal instability and carcinogenesis.

What is the nature of the physical interaction between BRIP1 and BRCA1?

BRIP1 interacts with BRCA1 through binding to the BRCT (BRCA1 C-Terminus) domains of BRCA1 . This interaction is phosphorylation-dependent, specifically requiring the phosphorylation of BRIP1 at Serine 990 . The BRIP1-BRCA1 complex formation is cell cycle-regulated, with phosphorylation occurring at specific cell cycle phases to facilitate timely DNA repair and checkpoint control . Experimental approaches to study this interaction include co-immunoprecipitation assays with phosphorylation-specific antibodies, proximity ligation assays, and FRET-based interaction studies in living cells.

How do mutations affect the BRIP1-BRCA1 interaction and its functional consequences?

Mutations that disrupt the BRIP1-BRCA1 interaction domain have significant functional consequences. For example, the c.2992-2995delAAGA mutation, identified in a woman with early-onset breast cancer, causes a frameshift that disrupts the BRCA1-binding domain and creates a premature stop codon . Functional analysis showed that this truncation interferes with both the stability of the protein and its ability to interact with BRCA1 . The mutant protein has an estimated half-life of approximately 1 hour compared to 5 hours for wild-type BRIP1 . Loss of the wild-type BRIP1 allele with retention of the mutated one in tumor tissue further supports its role in tumor suppression . These findings highlight the importance of maintaining the BRIP1-BRCA1 interaction for proper DNA repair function.

What experimental approaches are most effective for studying the BRIP1-BRCA1 interaction?

Several experimental approaches are effective for studying the BRIP1-BRCA1 interaction. Co-immunoprecipitation analysis is particularly valuable, as demonstrated in studies of novel BRIP1 mutations . When designing such experiments, it's crucial to include phosphatase inhibitors in all buffers to preserve the phosphorylation-dependent interaction. Using antibodies targeting different regions of both proteins can provide complementary information, especially when studying truncation mutants. For recombinant protein studies, in vitro transcription and translation reactions can be programmed with wild-type and mutant plasmids, followed by Western blotting analysis using antibodies against BRIP1 or epitope tags . Cell-based assays examining co-localization of BRIP1 and BRCA1 at DNA damage sites provide functional insights into this interaction in a physiological context.

What is the evidence linking BRIP1 mutations to breast cancer susceptibility?

Several lines of evidence link BRIP1 mutations to breast cancer susceptibility. Studies have identified truncating mutations in BRIP1 in breast cancer patients, particularly those with early-onset disease . The c.2992-2995delAAGA mutation, for instance, was found in a woman with early-onset breast cancer . This four-nucleotide deletion causes a frameshift that disrupts the BRCA1-binding domain and creates a premature stop codon . Furthermore, BRIP1 maps to chromosome 17q22 near the BRCA1 locus, a region that frequently shows allelic losses in breast carcinomas even when BRCA genes are wild-type, suggesting BRIP1 may be an additional breast cancer susceptibility gene . These findings collectively support BRIP1's role as a low-penetrance breast cancer predisposing gene.

How does loss of heterozygosity at the BRIP1 locus contribute to tumorigenesis?

Loss of heterozygosity (LOH) at the BRIP1 locus is a significant event in tumorigenesis, particularly in patients carrying a germline BRIP1 mutation. Analysis of breast tumor tissue from patients with BRIP1 mutations has revealed loss of the wild-type BRIP1 allele with retention of the mutant allele . This classic "second hit" pattern, demonstrated through techniques such as laser-capture microdissection of tumor specimens followed by DNA extraction and sequencing, provides strong evidence for BRIP1's tumor-suppressive function . The loss of functional BRIP1 compromises DNA repair mechanisms, leading to genomic instability and the accumulation of additional mutations that drive cancer progression.

What methodologies are recommended for detecting BRIP1 mutations in clinical samples?

For detecting BRIP1 mutations in clinical samples, several complementary methodologies are recommended. Initial screening can be performed using Single Strand Conformation Polymorphism (SSCP) analysis, where PCR products showing altered migration patterns are subsequently sequenced . Direct bidirectional sequencing using dye-terminator chemistry remains the gold standard for mutation confirmation . For comprehensive analysis, all coding exons and flanking intronic sequences should be amplified by PCR, with products typically ranging between 214 and 380 base pairs . Modern approaches may incorporate next-generation sequencing panels that include BRIP1 alongside other DNA repair genes. For functional characterization of identified variants, researchers should consider generating expression constructs for both wild-type and mutant proteins to assess effects on stability, localization, and protein-protein interactions .

What are the optimal applications for BRIP1 recombinant monoclonal antibodies in research?

BRIP1 recombinant monoclonal antibodies are versatile tools with several optimal research applications. Western blotting (WB) allows detection of BRIP1 protein in cell or tissue lysates, providing information about expression levels and potential isoforms or post-translational modifications . Immunocytochemistry/immunofluorescence (ICC/IF) enables visualization of BRIP1's subcellular localization and potential co-localization with interaction partners like BRCA1 . Immunohistochemistry on paraffin-embedded sections (IHC-P) facilitates examination of BRIP1 expression patterns in tissue specimens, including clinical samples . Immunoprecipitation (IP) is particularly valuable for studying protein-protein interactions and isolating BRIP1-containing complexes . When selecting antibodies for these applications, researchers should consider epitope location, especially when studying truncation mutants that may lack C-terminal regions.

How can researchers validate the specificity of BRIP1 recombinant monoclonal antibodies?

Validating BRIP1 recombinant monoclonal antibodies requires a multi-faceted approach. Western blotting should be performed with positive controls (cells known to express BRIP1) alongside negative controls (BRIP1 knockout cells or siRNA-mediated knockdown). Multiple antibodies targeting different epitopes of BRIP1 should yield consistent results in terms of molecular weight (approximately 140.9 kDa for the canonical protein) . For N-terminal antibodies like ab151509, recognition of recombinant fragments (e.g., amino acids 50-300) can be confirmed . Immunostaining should show the expected nuclear and cytoplasmic localization pattern for BRIP1 . Additionally, appropriate controls should be included for each application, such as secondary antibody-only controls for immunofluorescence and isotype controls for immunoprecipitation. Cross-reactivity with related DEAH helicase family members should be assessed, particularly when studying novel cell types or organisms.

What technical challenges are associated with BRIP1 antibody-based experiments?

Several technical challenges must be addressed when conducting BRIP1 antibody-based experiments. First, the existence of multiple isoforms through alternative splicing necessitates careful epitope selection to ensure detection of all relevant protein variants . Second, post-translational modifications like phosphorylation and acetylation can affect antibody recognition, particularly if the epitope contains modification sites . Third, the relatively low abundance of endogenous BRIP1 in some cell types may require sensitive detection methods and optimization of extraction procedures. For experiments involving mutant BRIP1 proteins, the potential instability of these variants poses a challenge; for instance, the c.2992-2995delAAGA mutant has a significantly shorter half-life (approximately 1 hour) compared to wild-type BRIP1 (approximately 5 hours) . Finally, when studying BRIP1-BRCA1 interactions, preserving phosphorylation status is critical, necessitating the inclusion of appropriate phosphatase inhibitors throughout experimental procedures.

How can BRIP1 antibodies be utilized to study post-translational modifications?

BRIP1 antibodies can be strategically employed to study post-translational modifications through several approaches. Phosphorylation-specific antibodies targeting key sites, such as Serine 990 which mediates BRCA1 interaction, allow direct detection of these modified forms in different cellular contexts . For comprehensive analysis, immunoprecipitation using total BRIP1 antibodies followed by Western blotting with modification-specific antibodies (phospho-, acetyl-, etc.) can reveal the modification status under different conditions. Mass spectrometry analysis of immunoprecipitated BRIP1 provides an unbiased approach to identify novel modification sites. Two-dimensional gel electrophoresis combined with Western blotting can separate differently modified forms based on charge and mass. Additionally, antibodies recognizing total BRIP1 can be used in combination with phosphatase or deacetylase treatments to determine how these modifications affect protein stability, localization, and function.

What methodological approaches can detect conformational changes in BRIP1 during DNA damage response?

Detecting conformational changes in BRIP1 during DNA damage response requires sophisticated methodological approaches. Proximity ligation assays using antibodies against different BRIP1 domains can reveal changes in protein folding that alter the spatial relationship between epitopes. Fluorescence resonance energy transfer (FRET) between fluorescently labeled antibodies targeting different regions of BRIP1 provides a sensitive measure of conformational dynamics in living cells. Limited proteolysis followed by epitope-specific antibody detection can identify regions that become more or less accessible after DNA damage. Hydrogen-deuterium exchange mass spectrometry of immunopurified BRIP1 offers detailed structural information about which protein regions experience altered solvent exposure following damage. Additionally, comparing the immunoprecipitation efficiency of different domain-specific antibodies before and after DNA damage can provide insights into epitope accessibility changes reflecting conformational alterations.

How can researchers investigate the relationship between BRIP1 helicase activity and its role in G-quadruplex resolution?

Investigating the relationship between BRIP1 helicase activity and G-quadruplex resolution requires specialized experimental approaches. Helicase assays using purified recombinant BRIP1 (wild-type or mutant) and synthetic G-quadruplex substrates with different structural features can directly measure unwinding activity in vitro . Chromatin immunoprecipitation (ChIP) using BRIP1 antibodies followed by sequencing can identify genomic regions enriched for BRIP1 binding, which can be correlated with predicted G-quadruplex forming sequences. Cell-based assays measuring BRIP1 recruitment to G-rich regions (such as telomeres) after inducing replication stress provide functional insights. For mechanistic studies, site-directed mutagenesis of key helicase domain residues followed by functional rescue experiments can determine which aspects of BRIP1's enzymatic activity are essential for G-quadruplex resolution. Additionally, combining BRIP1 antibodies with G-quadruplex-specific antibodies in co-localization or proximity ligation assays can visualize these interactions in their cellular context.

Table 1: Common Applications and Specifications for BRIP1 Antibodies