FIGNL1 Antibody

What is the functional role of FIGNL1 in DNA repair mechanisms?

FIGNL1 is an essential component in homologous recombination (HR) repair pathways. It specifically interacts with RAD51 through its conserved RAD51 binding domain (FRBD) . Experiments have demonstrated that cells depleted of FIGNL1 show defective HR repair and increased sensitivity to DNA damaging agents such as camptothecin and ionizing radiation .

FIGNL1 appears to function in the RAD51-mediated strand invasion process during HR. The protein contains an AAA ATPase domain and is capable of modulating RAD51 nucleoprotein filaments. Mechanistically, FIGNL1 can dissociate RAD51 from DNA, suggesting a regulatory role in controlling RAD51 activity during the DNA repair process . This dissociation activity is ATP-dependent and involves direct interaction with the N-terminus of RAD51 .

What are the key structural domains of FIGNL1 and their functions?

FIGNL1 contains several critical domains that contribute to its function:

• RAD51 Binding Domain (FRBD): Located at residues 295-344 in human FIGNL1, this conserved region is essential for direct interaction with RAD51 . Within this domain:

  • Site 1 contains an FxxA motif that binds to RAD51 at the protomer interface

  • Site 2 includes an FVPP motif that can bind to RAD51 molecules within filaments

• N-terminal region (residues 1-120): This region is necessary and sufficient for FIGNL1 recruitment to DNA damage sites . It functions independently of the FRBD domain, allowing FIGNL1 to localize to DNA damage even when its interaction with RAD51 is disrupted.

• AAA ATPase domain: Located in the C-terminal portion of FIGNL1, this domain contains critical residues (including K447 and D500) involved in ATP hydrolysis . The ATPase activity is stimulated by RAD51, specifically through interaction with RAD51's N-terminus.

• Nuclear Localization Signal (NLS): Required for proper nuclear localization and interaction with FIRRM/FLIP, which stabilizes FIGNL1 .

Which proteins interact with FIGNL1 and what complexes does it form?

FIGNL1 forms functional complexes with several key proteins:

• RAD51: The central recombinase in HR repair. FIGNL1 directly binds to RAD51 through its FRBD domain . This interaction is essential for FIGNL1's function in HR repair.

• FIRRM/FLIP: Forms a complex with FIGNL1 that is essential for meiotic recombination. The FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin . Depletion of either FIGNL1 or FIRRM in mouse spermatocytes results in meiotic DSB repair failure .

• KIAA0146/SPIDR: Identified as a binding partner of FIGNL1, this scaffolding protein involved in DNA repair acts with FIGNL1 in HR repair . Unlike RAD51, the binding of KIAA0146 to FIGNL1 does not require the FRBD domain.

• MMS22L-TONSL complex: FIGNL1 interacts with both MMS22L and TONSL proteins . Since both MMS22L-TONSL and FIGNL1 modulate RAD51 activity in HR, these interactions may represent functional coordination in the DNA repair pathway.

• DMC1: In addition to RAD51, FIGNL1 can also interact with and disrupt DMC1-DNA associations, although with lower efficiency than RAD51 . This interaction is particularly relevant in meiotic recombination.

How does FIGNL1 regulate RAD51 filament dynamics during homologous recombination?

FIGNL1 plays a sophisticated role in regulating RAD51 filament dynamics through several mechanisms:

FIGNL1 directly dissociates RAD51 from DNA through an ATP-dependent mechanism . This activity involves the AAA ATPase domain of FIGNL1, which is stimulated by interaction with the N-terminus of RAD51. The process appears to involve several distinct steps:

• Recognition and binding: FIGNL1 recognizes RAD51 filaments through its FRBD domain. The FxxA motif (Site 1) preferentially binds to RAD51 at filament ends or gaps, while the FVPP motif (Site 2) can bind to RAD51 molecules within a filament .

• ATPase activation: Once bound, the RAD51 N-terminus stimulates FIGNL1's ATPase activity .

• Conformational changes: ATP hydrolysis likely drives conformational changes in FIGNL1 that physically disrupt RAD51's association with DNA.

• Filament dissolution: This results in the removal of RAD51 from DNA, potentially regulating the stability and processing of recombination intermediates.

Interestingly, while FIGNL1 can disrupt RAD51 filaments, it does not affect the initial loading of RAD51 onto ssDNA . This suggests a role in fine-tuning rather than preventing RAD51 activity, consistent with FIGNL1's function in promoting efficient HR repair rather than blocking it entirely.

What is the mechanism by which FIGNL1 contributes to meiotic recombination?

In meiotic recombination, FIGNL1 forms a complex with FIRRM that is essential for proper meiotic double-strand break (DSB) repair . The FIGNL1-FIRRM complex regulates both RAD51 and DMC1 recombinases, which are critical for homology search and strand invasion during meiotic recombination.

Key aspects of FIGNL1's role in meiosis include:

• Limiting excessive recombinase loading: The FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin, independent of SPO11-catalyzed DSB formation . This suggests a role in preventing inappropriate recombinase activity.

• Filament modification: Purified human FIGNL1ΔN alters RAD51/DMC1 nucleoprotein filament structure and inhibits strand invasion in vitro . This activity may help regulate the strand invasion process to ensure accurate homologous recombination.

• Promoting efficient processing: By regulating recombinase activity, FIGNL1-FIRRM promotes proficient strand invasion and proper processing of recombination intermediates .

Experimental evidence shows that depletion of FIGNL1 or FIRRM in mouse spermatocytes results in meiotic DSB repair failure and prevents full synapsis between homologous chromosomes . This underscores the critical role of this complex in ensuring proper meiotic recombination.

How does FIGNL1 function differ in BRCA2-deficient versus BRCA2-proficient cells?

FIGNL1 exhibits interesting functional relationships with BRCA2 that reveal insights into DNA repair pathway regulation:

This complex relationship suggests that FIGNL1 may play different roles depending on the status of BRCA2: in BRCA2-proficient cells, it promotes efficient HR by regulating RAD51 filament dynamics, while in BRCA2-deficient cells, its RAD51-dissociating activity might further impair the already compromised HR pathway.

What techniques are most effective for studying FIGNL1-RAD51 interactions?

Several complementary techniques have proven effective for investigating FIGNL1-RAD51 interactions:

• Co-immunoprecipitation (Co-IP): Effective for detecting protein-protein interactions in cell lysates. Researchers have successfully used this approach with EGFP-tagged FIGNL1 in FIGNL1-/- RPE1 cells to demonstrate interactions with RAD51, MMS22L, and TONSL .

• Direct binding assays with purified proteins: In vitro assays using purified components have been instrumental in defining the direct interaction between FIGNL1 and RAD51. These assays helped identify the FRBD domain and demonstrated that the FRBD domain alone can bind to RAD51 .

• Nuclease protection assays: These assays evaluate FIGNL1's ability to disrupt RAD51-DNA filaments by measuring protection of DNA from nuclease digestion . They have been used to assess both RAD51 and DMC1 filament disruption by FIGNL1.

• ATPase activity assays: Measuring ATP hydrolysis rates has demonstrated that RAD51, specifically its N-terminal region, stimulates FIGNL1's ATPase activity .

• Structural prediction using AlphaFold: Computational approaches like AlphaFold have been employed to predict the structural interactions between FIGNL1, RAD51, and other partners like MMS22L-TONSL .

• Microscopy techniques for co-localization: Immunofluorescence microscopy has been used to demonstrate co-localization of FIGNL1 with γH2AX foci at sites of DNA damage .

How can one generate and validate FIGNL1 mutants for functional studies?

Creating and validating FIGNL1 mutants requires careful design and multiple validation steps:

• Design strategy for functional mutants:

  • FRBD domain mutants: Delete residues 295-344 or introduce point mutations (e.g., F295E) in the FxxA motif to disrupt RAD51 binding

  • ATPase domain mutants: Introduce point mutations K447A+D500A to disrupt ATP hydrolysis

  • N-terminal region mutants: Delete residues 1-120 to prevent localization to DNA damage sites

  • NLS mutants: Disrupt the nuclear localization signal to prevent interaction with FIRRM/FLIP

• Expression systems:

  • Mammalian expression: Use shRNA-resistant constructs to express mutant versions while depleting endogenous FIGNL1

  • Bacterial expression: FIGNL1ΔN can be expressed in E. coli BL21(DE3) cells as an N-terminal 6xHistidine fusion with a C-terminal Strep-tag

• Functional validation approaches:

  • Interaction assays: Co-IP or direct binding assays to confirm disruption of specific protein interactions

  • Localization studies: Microscopy to verify nuclear localization and recruitment to DNA damage sites

  • HR reporter assays: Functional complementation experiments to assess HR repair efficiency

  • Sensitivity assays: Cell survival measurements after exposure to DNA damaging agents

  • Biochemical assays: ATPase activity measurements and nuclease protection assays

What controls are essential when using FIGNL1 antibodies for immunoprecipitation?

When performing immunoprecipitation experiments with FIGNL1 antibodies, several critical controls should be included:

• Negative controls:

  • IgG control: Use isotype-matched IgG to control for non-specific binding

  • FIGNL1 knockout/knockdown cells: Perform parallel IP in cells lacking FIGNL1 to identify non-specific bands

  • Peptide competition: Pre-incubate antibody with excess antigenic peptide to confirm specificity

• Positive controls:

  • Known interacting partners: Probe for established FIGNL1 interactors like RAD51, FIRRM, or KIAA0146/SPIDR

  • Domain-specific mutants: Include FRBD deletion mutants that should abolish RAD51 interaction but maintain other interactions like KIAA0146

• Experimental conditions to optimize:

  • DNA damage induction: Compare IP results with and without damage (e.g., IR treatment)

  • Cell cycle synchronization: FIGNL1 functions may be cell-cycle dependent

  • Nuclear extraction protocols: Ensure efficient extraction of nuclear proteins

  • Salt concentration: Titrate salt conditions to maintain specific interactions while reducing background

• Validation approaches:

  • Reciprocal IP: Confirm interactions by performing IP with antibodies against interacting partners

  • Domain mapping: Use truncated constructs to verify interaction domains

  • Mass spectrometry: Identify novel interacting proteins and confirm established ones

How can researchers distinguish between FIGNL1's roles in different DNA repair pathways?

Distinguishing FIGNL1's functions across different repair pathways requires strategic experimental design:

• Pathway-specific assays:

  • HR-specific reporter assays: DR-GFP or similar systems specifically measure HR efficiency

  • Meiosis-specific analysis: Assess FIGNL1 function in spermatocytes to specifically study meiotic recombination

  • Alternative pathway measurements: Include assays for non-homologous end joining (NHEJ) to determine pathway specificity

• Cell type considerations:

  • Germ cells vs. somatic cells: Compare FIGNL1 function between these cell types to distinguish meiotic vs. mitotic roles

  • Cancer vs. normal cells: Evaluate FIGNL1 function in BRCA2-deficient vs. proficient cells

• Temporal analysis:

  • Cell cycle synchronization: Analyze FIGNL1 function at specific cell cycle phases

  • Time-course experiments: Track FIGNL1 recruitment and function at different times after DNA damage

• Genetic approaches:

  • Epistasis analysis: Combine FIGNL1 depletion with knockdown of pathway-specific factors (e.g., BRCA2, RAD51 paralogs) to identify genetic interactions

  • Synthetic lethality screens: Identify contexts where FIGNL1 becomes essential

• Damage-specific approaches:

  • Use different damaging agents: Compare responses to IR (causes DSBs) vs. camptothecin (causes replication-associated breaks) vs. interstrand crosslinkers

What approaches can resolve contradictory phenotypes in different FIGNL1 experimental models?

Resolving contradictory phenotypes requires systematic investigation:

• Methodological standardization:

  • Knockout vs. knockdown: Compare complete knockout versus partial depletion phenotypes

  • Acute vs. chronic depletion: Use inducible systems to distinguish immediate versus adaptive responses

  • Expression level control: Ensure complementation experiments use physiological expression levels

• Domain-specific analysis:

  • Structure-function studies: Use the various FIGNL1 mutants (FRBD, ATPase, N-terminal) to determine which functions are responsible for specific phenotypes

  • Separation-of-function mutants: Design mutants that disrupt specific interactions while preserving others

• Context-dependent functions:

  • Cell type variation: Systematically compare FIGNL1 function across different cell types

  • Genetic background effects: Test FIGNL1 function in different genetic contexts (e.g., BRCA2-proficient vs. deficient)

  • DNA damage type: Compare phenotypes with different damage induction methods

• Interaction network mapping:

  • Comprehensive protein interaction studies: Identify cell-type specific interactors that might explain phenotypic differences

  • Post-translational modification analysis: Determine if FIGNL1 regulation differs between experimental systems

• Quantitative approaches:

  • Dose-response studies: Titrate FIGNL1 levels to identify threshold effects

  • Kinetic measurements: Analyze the timing of FIGNL1 recruitment and function

Why does FIGNL1 localize to DNA damage sites independently of RAD51 despite their functional interaction?

This apparent contradiction reveals important insights about FIGNL1's multifaceted functions:

• Distinct recruitment mechanisms: The N-terminal region of FIGNL1 (residues 1-120) is necessary and sufficient for FIGNL1 focus formation at DNA damage sites, while RAD51 binding occurs through the separate FRBD domain (residues 295-344) . This domain separation allows independent recruitment.

• Early arrival hypothesis: FIGNL1 may be recruited to damage sites before RAD51, possibly to prepare the chromatin environment or interact with early-arriving repair factors.

• Alternative interaction partners: FIGNL1 may be recruited through interactions with other damage-response proteins like KIAA0146/SPIDR , which could explain its BRCA2/RAD51-independent localization.

• Regulatory model: FIGNL1's independent localization may be a regulatory mechanism ensuring it is present at damage sites only when needed for RAD51 regulation, preventing inappropriate RAD51 filament disruption.

• Dual functionality: FIGNL1 may have functions at damage sites beyond RAD51 regulation, explaining why it would need recruitment mechanisms independent of RAD51.

Experimentally, this has been demonstrated by showing that FRBD deletion mutants, which cannot bind RAD51, still localize to DNA damage foci . This is distinctly different from other RAD51-interacting proteins like RAD51AP1, where deletion of the RAD51 binding domain abolishes both RAD51 interaction and damage site localization .

How can the seemingly contradictory roles of FIGNL1 in both promoting and inhibiting HR be reconciled?

The dual roles of FIGNL1 in HR can be reconciled through a nuanced understanding of recombination dynamics:

This complex regulatory role is supported by seemingly contradictory observations: FIGNL1 depletion impairs HR repair efficiency , yet FIGNL1 can dissociate RAD51 from DNA . The resolution lies in understanding that controlled and timely removal of RAD51 from specific DNA structures is as important for successful HR as proper RAD51 loading.

What are the most promising research areas for further understanding FIGNL1 function?

Several promising research directions emerge from current FIGNL1 knowledge:

• Structural biology approaches: Obtaining high-resolution structures of FIGNL1 in complex with RAD51 and other partners would provide mechanistic insights into how FIGNL1 regulates recombinase activity.

• Single-molecule studies: Real-time observation of FIGNL1 action on individual RAD51 filaments could reveal the dynamics and specificity of FIGNL1's regulatory function.

• Therapeutic targeting: Exploring FIGNL1 as a potential therapeutic target in BRCA2-deficient cancers, where FIGNL1 inhibition might restore some HR capacity.

• Meiosis-specific functions: Further investigation of FIGNL1-FIRRM in meiotic recombination could reveal insights into fertility disorders and meiotic chromosome segregation.

• Regulatory mechanisms: Understanding how FIGNL1 itself is regulated through post-translational modifications or protein interactions could reveal additional layers of DNA repair control.