RAD51B Human

What is RAD51B and what is its genomic location?

RAD51B is a gene located at chromosome 14q24.1 that encodes a member of the RAD51 protein family. This highly conserved protein is one of five RAD51 paralogs identified in vertebrates and exhibits central recombinase activity in mammalian cells . RAD51B functions as a known RAD51 paralog and plays a critical role in DNA repair mechanisms, particularly in homologous recombinational repair (HRR) of DNA double-strand breaks (DSBs) . The protein is evolutionarily conserved, highlighting its fundamental importance in cellular metabolism across species.

The RAD51 protein family members are essential components of DNA repair pathways, with RAD51B being specifically involved in the initial stages of homologous recombination. The conservation of this gene family throughout evolution underscores their critical function in maintaining genomic integrity, as mutations with high functional significance in these genes might be lethal . The sequence surrounding the putative ATP binding sites is highly conserved between chicken and human RAD51B proteins, although interestingly, the typically well-conserved GXXXXGKTQ motif in the Walker A box is changed to SXXXXGKTQ in the chicken sequence .

What are the primary functions of RAD51B in cellular processes?

RAD51B primarily functions in homologous recombinational repair (HRR) of DNA double-strand breaks, which is essential for maintaining genomic stability . The protein plays a crucial role in the formation of RAD51B nucleoprotein filaments, which represent the initial stage of HRR . Absence of RAD51B may disrupt this filament formation, potentially resulting in DNA mutations, rearrangements, and/or chromosomal loss . These genomic alterations can lead to various pathological conditions, including increased susceptibility to certain diseases.

Beyond its role in DNA repair, RAD51B has been identified as a promising candidate oncogene and potential biomarker for cancer diagnosis and prognosis . Functional studies have demonstrated that RAD51B, when complexed with RAD51C, exhibits single-stranded DNA (ssDNA) binding and ssDNA-stimulated ATPase activities . This complex acts as a mediator in recombination processes, facilitating the assembly of the RAD51-ssDNA nucleoprotein filament in vivo, which is essential for initiating homologous recombination . The mediator function becomes particularly important when RPA (Replication Protein A) competes with RAD51 for binding sites on ssDNA, as the RAD51B-RAD51C complex can partially overcome this suppressive effect .

How can RAD51B knockout models be generated for functional studies?

Generating RAD51B knockout models requires careful design of targeting vectors to disrupt the gene's function. Based on established methodologies, researchers can create RAD51B disruption constructs by first isolating a partial cDNA encoding the target gene. For instance, in chicken DT40 cells, two RAD51B disruption constructs (RAD51B-puro and RAD51B-bsr) were designed to replace the chicken RAD51B coding sequence for amino acids 137 to 173 with selection markers . These constructs contained ~1.8-kb and ~2.4-kb fragments at the RAD51B locus cloned in a pBS vector, with an artificially generated unique BamHI site between the fragments for inserting drug resistance cassettes .

The targeting vectors should be linearized before transfection (e.g., using NotI and SacI restriction enzymes) . After transfection, successful disruption of both RAD51B alleles can be verified through Southern and Northern blot analyses . It's important to validate the knockout by confirming the absence of functional protein expression. For comprehensive functional studies, researchers should also generate complementation models where human RAD51B is expressed in the knockout cells to rescue the phenotype and confirm the specificity of observed effects . This approach has been successfully used to demonstrate that high expression of human RAD51B in RAD51B−/− clones restored their growth rate to wild-type levels .

What methodologies are effective for studying RAD51B-RAD51C complex formation and activity?

Studying the RAD51B-RAD51C complex requires a combination of biochemical, biophysical, and functional approaches. To characterize the complex formation, researchers can use co-immunoprecipitation assays with antibodies specific to either RAD51B or RAD51C, followed by Western blot analysis to detect the partner protein. For more rigorous analysis, size exclusion chromatography can help determine whether the complex forms with a defined stoichiometry, which has been established as a stable stoichiometric complex in previous studies .

To investigate the biochemical activities of the RAD51B-RAD51C complex, researchers should assess its DNA binding properties using electrophoretic mobility shift assays (EMSAs) with single-stranded DNA substrates. ATPase activity can be measured using radioactive assays that track the hydrolysis of [γ-32P]ATP to ADP and inorganic phosphate . The functional significance of the complex in homologous recombination can be studied through in vitro recombination assays that measure DNA strand exchange activities. In these assays, researchers should include conditions with and without RPA to assess the mediator function of the RAD51B-RAD51C complex . The complex's ability to overcome RPA's inhibitory effect on RAD51-mediated DNA pairing and strand exchange provides important insights into its role in facilitating homologous recombination in vivo .

What is the association between RAD51B polymorphisms and rheumatoid arthritis?

RAD51B gene variants have been significantly associated with rheumatoid arthritis (RA) susceptibility in multiple populations. A comprehensive two-stage case-control study in Han Chinese individuals identified a common variant, rs911263, as significantly associated with RA disease status (P = 4.8 × 10−5, OR = 0.64) . This finding confirms previous associations reported in Korean and European populations, suggesting a conserved role for RAD51B in RA susceptibility across different ethnic groups . Importantly, the same SNP (rs911263) was also shown to be related to erosion, a clinical assessment of disease severity in RA (P = 2.89 × 10−5, OR = 0.52), indicating that this genetic variant may influence both disease onset and progression .

The protective effect of the C allele of rs911263 varies between populations, with an odds ratio of approximately 0.8 reported in previous meta-analyses, compared to 0.5-0.6 in the Han Chinese population . This difference might reflect population-specific genetic backgrounds or environmental factors that modify the effect of this variant. The mechanism through which RAD51B influences RA susceptibility remains unclear, but considering its fundamental role in DNA repair, it's possible that alterations in RAD51B function could affect immune cell development or responses to oxidative stress, both of which are implicated in RA pathogenesis. The consistent association of rs911263 with RA across multiple studies and populations significantly reduces the likelihood that this is merely a false positive signal due to confounding factors .

How should researchers design genetic association studies to investigate RAD51B in disease contexts?

Designing effective genetic association studies for RAD51B requires careful consideration of several key factors. First, researchers should conduct a comprehensive tag SNP selection process to ensure adequate coverage of common genetic variations in the target population. As demonstrated in the Han Chinese RA study, this process can involve searching for all SNPs with a minor allele frequency (MAF) ≥ 0.05 in the appropriate reference database (e.g., 1000-genomes CHB database) . Applying criteria such as MAF ≥ 0.05 with pair-wise tagging and r² ≥ 0.5 can help identify representative tag SNPs covering the entire RAD51B region .

Sample collection and genotyping methods should be standardized and well-documented. For instance, peripheral venous blood samples can be collected in plain tubes, with genomic DNA isolated according to manufacturer's protocols . High-throughput genotyping platforms, such as the Sequenom MassARRAY with iPLEX GOLD chemistry, provide reliable results for large-scale studies . Quality control measures should include assessing call rates, Hardy-Weinberg equilibrium, and using duplicate samples to verify genotyping accuracy.

For data analysis, researchers should employ bioinformatic tools to identify SNPs in strong linkage disequilibrium with significant variants. Web-based population genetics software like SNAP can help identify ungenotyped SNPs that are in strong LD with significant SNPs in the study population . To predict the potential functional significance of identified variants, especially for intronic/synonymous SNPs, databases such as RegulomeDB can annotate SNPs with known and predicted regulatory elements . Additionally, protein-protein interaction network databases like STRING can help investigate the network neighbors of RAD51B, providing insights into its functional relationships and potential disease mechanisms .

How does RAD51 overexpression affect cells with RAD51B deficiency?

Overexpression of RAD51 has been shown to suppress recombination defects in cells with RAD51B deficiency. In studies with chicken DT40 cells where RAD51B was knocked out, overexpression of human RAD51 significantly complemented various defects observed in these cells . Specifically, RAD51 overexpression restored resistance to mitomycin C (MMC), cisplatin, and X-ray exposure, and reduced genomic instability in RAD51B−/− cells . Interestingly, this complementation did not fully restore homologous integration events, suggesting that while RAD51 overexpression largely ameliorates HRR-deficiency in the absence of RAD51B, it cannot completely compensate for all functions of this paralog .

What is the role of the RAD51B-RAD51C complex in mediating homologous recombination?

The RAD51B-RAD51C complex serves as a crucial mediator in homologous recombination by facilitating the assembly of the RAD51-ssDNA nucleoprotein filament, which is essential for initiating the homologous DNA pairing and strand exchange process . While Replication Protein A (RPA) enhances RAD51-catalyzed DNA joint formation by removing secondary structures in the ssDNA substrate, it can also compete with RAD51 for binding to the substrate, which suppresses the reaction efficiency . The RAD51B-RAD51C complex helps overcome this competitive inhibition by RPA, partially alleviating the suppressive effect and promoting efficient recombination .

The complex exhibits intrinsic biochemical activities that are relevant to its mediator function. It possesses ssDNA binding capacity, allowing it to interact with the DNA substrate, and ssDNA-stimulated ATPase activity, which may provide energy for its function in the recombination process . The mechanism through which RAD51B-RAD51C promotes RAD51 filament formation likely involves direct protein-protein interactions with both RAD51 and RPA, potentially modifying their binding affinities or conformations to favor RAD51 assembly on the ssDNA.

This mediator function of the RAD51B-RAD51C complex is analogous to that of the Saccharomyces cerevisiae Rad55-Rad57 complex, highlighting evolutionary conservation of this mechanism . The importance of this function is underscored by the fact that deficiencies in RAD51 paralogs, including RAD51B, lead to recombination defects and increased sensitivity to DNA-damaging agents, which can be partially rescued by RAD51 overexpression . Understanding the precise molecular mechanisms by which the RAD51B-RAD51C complex facilitates RAD51 filament assembly remains an active area of research with implications for cancer biology and targeted therapies.

What cellular phenotypes are observed in RAD51B-deficient models?

RAD51B-deficient cellular models exhibit several distinct phenotypes that reflect the crucial role of this protein in DNA repair and genomic stability. In chicken DT40 cells with RAD51B knockout, a significantly lower proliferation rate was observed compared to wild-type cells . This growth defect was restored to wild-type levels when human RAD51B was expressed in the knockout cells, confirming the specificity of the phenotype . Flow cytometric analysis revealed that RAD51B−/− cultures contained more dead cells than wild-type cultures, indicating that the reduced proliferation rate is likely caused by an elevated rate of cell death rather than a lengthening of the cell cycle .

Chromosome analysis in RAD51B-deficient cells demonstrated a marked increase in chromosomal aberrations compared to wild-type cells. As shown in the following data table from studies in DT40 cells:

This table demonstrates that RAD51B−/− cells exhibited a significantly higher frequency of chromosomal aberrations (15 ± 3.1 per cell) compared to wild-type cells (1.3 ± 0.9 per cell) . The most common aberrations were chromatid and chromosome gaps. Importantly, expression of human RAD51 in RAD51B−/− cells completely suppressed these chromosomal aberrations, restoring the frequency to wild-type levels . Additionally, RAD51B−/− cells showed a reduced rate of intragenic homologous recombination repair, with the calculated Ig gene conversion rate decreasing from 8.3 × 10−4 in wild-type cells to 3.5 × 10−4 in RAD51B−/− cells .

What are the optimal methods for detecting and analyzing RAD51B expression and function in human samples?

Analyzing RAD51B expression and function in human samples requires a multi-faceted approach combining molecular, cellular, and functional techniques. For transcript analysis, quantitative real-time PCR (qRT-PCR) using specific primers for RAD51B is a reliable method to measure mRNA expression levels. RNA sequencing can provide more comprehensive information, including potential splice variants. At the protein level, Western blotting with validated antibodies against RAD51B remains the gold standard for quantification, while immunohistochemistry or immunofluorescence can reveal the spatial distribution of the protein in tissue samples.

For functional analyses, evaluating the formation of RAD51B foci in response to DNA damage provides valuable insights. Recent studies have successfully analyzed RAD51 foci, along with BRCA1 and FANCD2 foci, in sporadic breast cancer biopsies treated with X-rays ex vivo . The absence of such foci was closely correlated with likely defects in the BRCA1 pathway, demonstrating the utility of this approach in analyzing primary tumors . This method could be adapted for RAD51B analysis by using specific antibodies against RAD51B.

Genetic screening for RAD51B variants can be performed using targeted sequencing, whole-exome sequencing, or genome-wide association studies depending on the research question. When examining the potential functional impact of identified variants, in silico prediction tools such as RegulomeDB can be used to annotate SNPs with known and predicted regulatory elements . For variants in coding regions, functional assays should be employed to directly assess their impact on protein function. These may include complementation studies in RAD51B-deficient cells or biochemical assays measuring the variant protein's ability to bind DNA, interact with RAD51C, and stimulate homologous recombination. Combining these molecular and functional approaches provides a comprehensive assessment of RAD51B status in human samples.

What is the potential of targeting RAD51B in cancer therapeutics?

The potential for targeting RAD51B in cancer therapeutics stems from its critical role in homologous recombination repair (HRR) of DNA double-strand breaks and its identification as a promising candidate oncogene and biomarker for cancer diagnosis and prognosis . Cancer cells often exhibit genomic instability and rely heavily on DNA repair mechanisms for survival, making components of these pathways attractive therapeutic targets. As RAD51B functions in a complex with RAD51C to mediate RAD51 filament formation during HRR, inhibiting this complex could potentially sensitize cancer cells to DNA-damaging agents or exploit synthetic lethality in tumors with specific genetic backgrounds.

Research has shown that overexpression of RAD51 can suppress recombination defects in cells with deficiencies in RAD51 paralogs or other HRR genes . This suggests that cancer cells might upregulate RAD51 expression as a compensatory mechanism when components of the HRR pathway, such as RAD51B, are dysfunctional. If so, combining inhibitors targeting RAD51B-RAD51C complex with agents that prevent RAD51 overexpression could potentially enhance therapeutic efficacy. Additionally, understanding the regulatory mechanisms controlling RAD51B expression, including transcriptional regulators and post-translational modifications, could reveal new therapeutic targets.

To advance this research direction, further investigation is needed to determine which tumors might be most vulnerable to RAD51B targeting. Key questions include: (i) Do tumors that overexpress RAD51 have suppressed HRR defects and TP53 mutations? (ii) If epigenetic silencing is involved in inactivating HRR functions, can these silencing events be reversed by drugs, and would this reversal be lethal to cancer cells with high levels of RAD51 protein? Answering these questions could guide the development of targeted therapies based on RAD51B biology and identify patient populations most likely to benefit from such approaches.

How can advanced genomic and proteomic technologies enhance our understanding of RAD51B networks?

Advanced genomic and proteomic technologies offer powerful approaches to unravel the complex networks involving RAD51B. CRISPR-Cas9 screens can systematically identify synthetic lethal interactions with RAD51B deficiency or overexpression, revealing potential therapeutic targets and pathway dependencies. Such screens could be performed in various cancer cell lines to identify context-dependent interactions that might be exploited for targeted therapies. Complementing these functional genomic approaches, proteomic methods like proximity labeling (BioID or APEX) can map the protein interaction network of RAD51B in living cells, potentially identifying novel binding partners beyond the known interaction with RAD51C.

Phosphoproteomic analysis following DNA damage could reveal how RAD51B is regulated by post-translational modifications and how it contributes to signaling cascades in the DNA damage response. Similarly, chromatin immunoprecipitation followed by sequencing (ChIP-seq) could identify genomic regions where RAD51B is recruited after DNA damage, providing insights into its genome-wide distribution and potential preferences for specific chromatin contexts or DNA sequences. Integration of these datasets with existing protein-protein interaction networks, such as those available through databases like STRING, would provide a comprehensive view of RAD51B's functional relationships .

For analyzing complex genetic interactions, genome-wide association studies incorporating RAD51B variants could identify modifier genes that influence disease susceptibility or response to therapy. The large-scale genomic and clinical data available through resources like The Cancer Genome Atlas (TCGA) can be leveraged to correlate RAD51B expression or mutation status with clinical outcomes across cancer types. These integrative approaches, combining multiple technological platforms and data types, hold great promise for advancing our understanding of RAD51B's role in DNA repair, cancer biology, and other disease contexts, potentially leading to new diagnostic markers and therapeutic strategies.