ZRANB3 Antibody

What is ZRANB3 and what cellular functions does it perform?

ZRANB3 is a 1079 amino acid nuclear protein belonging to the SNF2/RAD54 helicase family with a molecular weight of approximately 123.2 kDa. It functions as a structure-specific ATP-dependent endonuclease critical for maintaining genomic stability during DNA replication .

ZRANB3's primary functions include:

• Cleaving branched DNA structures with unusual polarity

• Processing stalled replication forks

• Facilitating replication-associated DNA repair

• Generating accessible 3'-OH groups in the template of the leading strand

The protein localizes to sites of active DNA synthesis and is rapidly recruited to DNA damage sites, with recruitment kinetics similar to those of PCNA and FEN1 . ZRANB3 interacts with key components of the DNA replication machinery, including PCNA and the MCM helicase complex, indicating its integral role in DNA replication processes .

Why are ZRANB3 antibodies important tools in DNA repair research?

ZRANB3 antibodies are invaluable tools for investigating the spatial and temporal regulation of DNA damage response pathways. They allow researchers to:

• Track ZRANB3 recruitment to sites of DNA damage through immunofluorescence

• Identify protein-protein interactions through co-immunoprecipitation assays

• Validate knockdown efficiency in functional studies of ZRANB3

• Distinguish between normal and aberrant ZRANB3 expression or localization patterns

When selecting antibodies for DNA repair research, considerations should include:

• Specificity for the isoform(s) of interest (ZRANB3 has 5 different isoforms from alternative splicing)

• Cross-reactivity with proteins from model organisms (human, mouse, rat, etc.)

• Compatibility with fixation protocols used in DNA damage studies

• Validated application for techniques such as Western blotting, immunocytochemistry, or immunoprecipitation

What structural domains should researchers target when selecting ZRANB3 antibodies?

The efficacy of ZRANB3 antibodies depends largely on which structural domain they target. ZRANB3 contains several distinct functional domains:

For studying ZRANB3's replication functions, antibodies targeting the PIP-box region (containing the PCNA-interacting motif) may be particularly useful, as mutations in this region (Q519A, F525A, and F526A) have been shown to abrogate ZRANB3's interaction with PCNA . For investigating nuclease activity, antibodies targeting the C-terminal HNH domain would be more appropriate. The C-terminal region (aa 1029-1079) is also commonly targeted in commercial antibodies .

What are the optimal methods for detecting ZRANB3 in replication foci?

To effectively detect ZRANB3 at replication foci, researchers should consider the following methodological approaches:

Immunofluorescence with BrdU co-labeling:

• Pulse-label cells with BrdU to mark sites of active DNA synthesis

• Fix cells using paraformaldehyde (typically 4%) for 10-15 minutes

• Permeabilize with 0.2% Triton X-100

• Co-stain with anti-ZRANB3 antibody and anti-BrdU antibody

• Include PCNA co-staining to validate replication foci

This approach has been validated in previous studies showing YFP-ZRANB3 colocalizing with sites of BrdU incorporation . For optimal results, antibodies specifically validated for immunocytochemistry (ICC) or immunofluorescence (IF) applications should be used .

Live-cell imaging approach:

• Generate stable cell lines expressing fluorescently tagged ZRANB3 (e.g., YFP-ZRANB3)

• Use DNA-intercalating dyes that are safe for live cells to mark replication sites

• Employ confocal microscopy with environmental controls (temperature, CO2)

• Capture time-lapse images to monitor ZRANB3 dynamics at replication sites

This approach allows for real-time tracking of ZRANB3 recruitment to replication sites or DNA damage areas, similar to methods used to demonstrate ZRANB3 recruitment to laser-induced DNA damage within minutes .

How can researchers use ZRANB3 antibodies to study DNA damage response?

ZRANB3 antibodies are powerful tools for investigating the protein's role in the DNA damage response through several experimental approaches:

Microirradiation studies:

• Transfect cells with fluorescently tagged ZRANB3 or prepare for immunostaining

• Induce localized DNA damage using laser microirradiation

• Track ZRANB3 recruitment to damaged sites over time through live imaging or fixed timepoints

• Compare recruitment kinetics with known DNA damage response factors (e.g., PCNA, FEN1)

Previous research has shown that ZRANB3 is recruited to microirradiated stripes within the first minute following DNA damage induction, with kinetics similar to PCNA and FEN1 but slower than ALC1 .

DNA damage sensitivity assays:

• Establish ZRANB3 knockdown or knockout cell lines

• Validate knockdown efficiency using ZRANB3 antibodies in Western blotting

• Expose cells to various DNA-damaging agents (MMS, camptothecin, hydroxyurea, etc.)

• Assess survival, cell cycle progression, and DNA damage marker accumulation

ZRANB3-deficient cells have demonstrated sensitivity to damage induced by methyl methanesulfonate (MMS) but not to camptothecin, hydroxyurea, H₂O₂, or UV radiation .

Co-immunoprecipitation after damage:

• Treat cells with DNA-damaging agents

• Perform immunoprecipitation using ZRANB3 antibodies

• Analyze changes in ZRANB3-associated proteins following damage

• Focus on interactions with known replication and repair factors (PCNA, MCM complex)

This approach can reveal how ZRANB3's protein interactions are modulated in response to different types of DNA damage.

What experimental approaches can reveal ZRANB3's interactions with the replication machinery?

To elucidate ZRANB3's interactions with replication machinery components, researchers can employ several methodological approaches:

Co-immunoprecipitation (Co-IP):

• Prepare cell lysates under conditions that preserve protein-protein interactions

• Immunoprecipitate ZRANB3 using validated antibodies

• Analyze precipitates for replication factors such as PCNA and MCM complex components

• Verify interactions through reciprocal Co-IPs (using antibodies against the interacting partners)

This approach has successfully demonstrated that ZRANB3 immunocomplexes contain PCNA and several MCM subunits (MCM3, MCM4, and MCM7) .

Proximity ligation assay (PLA):

• Fix cells and permeabilize according to standard immunostaining protocols

• Incubate with primary antibodies against ZRANB3 and a potential interacting partner

• Apply species-specific PLA probes and perform ligation and amplification

• Visualize interaction signals as distinct fluorescent spots

PLA provides in situ evidence of protein-protein interactions within 40 nm, offering spatial information about where in the cell ZRANB3 interactions occur.

PCNA interaction analysis using PIP-box mutants:

• Generate ZRANB3 constructs with mutated PIP-box residues (e.g., Q519A, F525A, F526A)

• Express wild-type and mutant constructs in appropriate cell lines

• Perform Co-IP experiments using ZRANB3 antibodies

• Compare PCNA binding between wild-type and mutant proteins

This targeted approach has confirmed that PIP-box mutations abrogate the interaction between ZRANB3 and PCNA, validating the specific domain responsible for this interaction .

How do the different isoforms of ZRANB3 affect antibody selection and experimental design?

ZRANB3 undergoes alternative splicing to generate five different isoforms, which presents both challenges and opportunities in research design . Strategic antibody selection can help address these complexities:

Isoform-specific experimental considerations:

• Identify the specific isoform(s) relevant to your research question

• Select antibodies that either:

  • Recognize all isoforms (targeting conserved regions) for global ZRANB3 studies

  • Target isoform-specific regions for differential analysis

• Validate antibody specificity using overexpression systems with individual isoforms

• Consider the tissue or cell type distribution of different isoforms

Methodological approaches for isoform discrimination:

• Western blotting with high-resolution SDS-PAGE to separate closely migrating isoforms

• Immunoprecipitation followed by mass spectrometry to identify specific isoforms

• RT-PCR analysis in parallel with protein detection to correlate isoform expression patterns

• Use of isoform-specific siRNAs to validate antibody specificity

When designing knockdown or knockout experiments, researchers should consider whether their approach will affect all or only specific ZRANB3 isoforms, as this may influence the interpretation of phenotypic outcomes.

What are the challenges in studying ZRANB3's ATP-dependent endonuclease activity?

Investigating ZRANB3's unique ATP-dependent endonuclease activity presents several methodological challenges that researchers must address:

Enzymatic activity assay considerations:

• Substrate preparation: ZRANB3 cleaves branched DNA structures with unusual polarity, requiring carefully designed DNA substrates

• ATP dependency: Assays must include appropriate ATP concentrations and controls without ATP

• Buffer optimization: Endonuclease activity is sensitive to salt concentrations, pH, and divalent cations

• Activity isolation: Separating ZRANB3's ATPase activity from its nuclease function requires targeted mutational analysis

Experimental approaches for nuclease activity characterization:

• In vitro cleavage assays using purified ZRANB3 and synthetic branched DNA substrates

• Testing structure specificity with a panel of different DNA structures (forks, Holliday junctions, bubbles)

• Analyzing cleavage products by high-resolution gel electrophoresis or sequencing

• Comparing wild-type activity with HNH domain mutants to confirm the catalytic center

The unusual polarity of ZRANB3's nuclease activity, which generates accessible 3'-OH groups in the template of the leading strand, makes it distinct from other structure-specific endonucleases and requires careful experimental design to accurately characterize .

How can researchers distinguish between ZRANB3's various functions at stalled replication forks?

ZRANB3 exhibits multiple functions at stalled replication forks, including ATP-dependent DNA translocation, structure-specific nuclease activity, and protein scaffold roles. Distinguishing between these functions requires sophisticated experimental approaches:

Domain-specific mutant analysis:

• Generate ZRANB3 constructs with targeted mutations in:

  • ATPase domain (to disable motor function)

  • HNH domain (to disable nuclease activity)

  • PIP-box (to prevent PCNA interaction)

  • NZF domain (to prevent K63-polyubiquitin binding)

• Express these mutants in ZRANB3-depleted cells

• Assess which cellular functions are rescued by each mutant

• Correlate functional rescue with specific biochemical activities

Fork processing analysis techniques:

• DNA fiber analysis to measure replication fork progression and restart

• Electron microscopy to visualize replication fork structures

• iPOND (isolation of Proteins On Nascent DNA) to identify ZRANB3-dependent protein recruitment

• 2D gel electrophoresis to analyze replication intermediates

Temporal dissection of ZRANB3 activities:

• Use inducible systems for rapid protein inactivation or recruitment

• Monitor replication fork markers at different time points after fork stalling

• Correlate changes in fork structure with ZRANB3's recruitment kinetics

• Employ live-cell imaging with fluorescently tagged replication and repair factors

These approaches can help determine whether ZRANB3's primary role at stalled forks is structural remodeling through its ATPase activity, nucleolytic processing via its endonuclease function, or serving as a scaffold to recruit additional repair factors.

What are the common pitfalls when using ZRANB3 antibodies in immunoprecipitation experiments?

Immunoprecipitation (IP) of ZRANB3 can be challenging due to its nuclear localization, relatively low abundance, and complex domain structure. Researchers should be aware of these common pitfalls:

Technical challenges and solutions:

Methodological optimizations:

• Lysis buffer selection: Use buffers containing 150-300 mM NaCl, 0.5-1% NP-40 or Triton X-100, with phosphatase and protease inhibitors

• Pre-clearing lysates: Incubate with protein A/G beads without antibody to reduce non-specific binding

• Antibody incubation time: Extend to overnight at 4°C for maximal binding

• Washing stringency: Balance between removing non-specific interactions while preserving specific ones

For co-IP studies investigating ZRANB3's interactions with replication machinery components like PCNA and MCM complex subunits, consider the cell cycle phase and DNA damage status, as these factors significantly influence interaction dynamics .

How can researchers validate ZRANB3 antibody specificity in their experimental systems?

Ensuring antibody specificity is crucial for obtaining reliable results when studying ZRANB3. A comprehensive validation strategy should include:

Antibody validation approaches:

• Western blot analysis comparing:

  • Wild-type vs. ZRANB3 knockdown/knockout cells

  • Tissue/cells known to express or not express ZRANB3

  • Detection of expected molecular weight (123.2 kDa for canonical form)

• Immunoprecipitation followed by mass spectrometry

• Immunofluorescence patterns compared with:

  • Fluorescently tagged ZRANB3 expression

  • siRNA knockdown controls

• Peptide competition assays to confirm epitope specificity

Critical controls for specific applications:

• For immunofluorescence:

  • Include secondary antibody-only controls

  • Compare replication foci staining patterns with established markers

  • Verify colocalization with PCNA or BrdU at replication sites

• For Western blotting:

  • Include positive control lysates from cells overexpressing ZRANB3

  • Verify band disappearance following siRNA treatment

  • Confirm expected shifts for tagged versions of the protein

• For immunoprecipitation:

  • Include IgG control precipitations

  • Verify enrichment of known interacting partners (PCNA, MCM subunits)

  • Confirm loss of specific interactions with appropriate mutants (e.g., PIP-box mutants)

What approaches help optimize ZRANB3 detection in different subcellular compartments?

ZRANB3 primarily localizes to the nucleus but shows dynamic distribution patterns during different cell cycle phases and in response to DNA damage. Optimizing detection in various subcellular compartments requires tailored approaches:

Subcellular fractionation techniques:

• Nuclear-cytoplasmic separation using gentle detergents (0.1% NP-40)

• Chromatin fractionation to separate soluble nuclear from chromatin-bound ZRANB3

• Nucleolar isolation to examine potential roles in ribosomal DNA maintenance

• Replication fork isolation using iPOND (isolation of Proteins On Nascent DNA)

Immunofluorescence optimization strategies:

• Fixation method selection:

  • Paraformaldehyde (2-4%) preserves protein-protein interactions

  • Methanol fixation enhances accessibility to some nuclear epitopes

  • Consider dual fixation for optimal results

• Permeabilization optimization:

  • Triton X-100 (0.1-0.5%) for general nuclear accessibility

  • Digitonin (10-50 μg/ml) for selective plasma membrane permeabilization

• Antigen retrieval methods:

  • Microwave or pressure cooker treatment in citrate buffer (pH 6.0)

  • Limited protease digestion to expose masked epitopes

• Signal amplification techniques:

  • Tyramide signal amplification for low-abundance detection

  • Multiple secondary antibody layers with careful washing

For studying ZRANB3's dynamic recruitment to DNA damage sites or replication forks, combining these techniques with cell synchronization or localized damage induction can provide temporal and spatial resolution of ZRANB3 activities .

How does ZRANB3 research contribute to our understanding of genome stability?

Research on ZRANB3 provides critical insights into mechanisms maintaining genome stability during DNA replication:

Key contributions to genome stability understanding:

• Replication fork protection: ZRANB3's structure-specific endonuclease activity helps process damaged replication forks, preventing fork collapse and genomic instability

• DNA damage tolerance: ZRANB3 facilitates replication through damaged DNA templates

• PCNA-mediated repair coordination: ZRANB3's interaction with PCNA connects it to the central hub of replication-coupled repair processes

• Structure-specific DNA processing: The unusual polarity of ZRANB3's nuclease activity creates accessible 3'-OH groups specifically in the leading strand template, providing insight into strand-specific repair mechanisms

Methodological approaches for studying ZRANB3's role in genome stability:

• Chromosomal aberration analysis in ZRANB3-deficient cells

• Sister chromatid exchange frequency measurements

• Micronuclei formation quantification following genotoxic stress

• DNA fiber analysis to measure fork stalling, collapse, and restart events

Research indicates that ZRANB3-deficient cells show increased sensitivity to MMS-induced DNA damage, highlighting its importance in specific DNA damage response pathways .

What are emerging roles for ZRANB3 beyond its known functions in DNA repair?

While ZRANB3's role in DNA repair and replication is well-established, emerging research suggests additional functions worthy of investigation:

Potential novel ZRANB3 functions:

• Transcriptional regulation: SNF2 family proteins often function in chromatin remodeling

• R-loop processing: Structure-specific endonucleases may resolve transcription-replication conflicts

• Telomere maintenance: Specialized replication structures require dedicated processing

• Mitochondrial DNA stability: Nuclear-encoded repair factors may function in organelles

Methodological approaches for exploring novel functions:

• ChIP-seq analysis to identify potential transcriptional regulation sites

• DRIP-seq (DNA-RNA Immunoprecipitation) to examine R-loop processing

• Proteomic profiling of ZRANB3 interactors in different cellular compartments

• Conditional knockout models to examine tissue-specific phenotypes

Researchers investigating these potential functions should consider developing highly specific experimental systems that can distinguish these activities from ZRANB3's established roles in replication and repair.

How might ZRANB3 function as a therapeutic target in DNA repair disorders?

Understanding ZRANB3's role in DNA repair has potential implications for therapeutic approaches in cancer and genetic disorders:

Therapeutic relevance considerations:

• Synthetic lethality: ZRANB3 inhibition might selectively kill cancer cells with defects in complementary repair pathways

• Replication stress targeting: Cancers with elevated replication stress may depend on ZRANB3 function

• Combination therapy: ZRANB3 inhibition could sensitize cells to existing chemotherapeutics or radiation

• Biomarker potential: ZRANB3 expression or activity may predict therapy response

Experimental approaches for therapeutic development:

• Small molecule screening for inhibitors of ZRANB3's ATPase or nuclease activities

• Structure-based drug design targeting the ATP-binding pocket or HNH domain

• Peptide inhibitors disrupting ZRANB3-PCNA interaction via the PIP-box

• PROTAC (Proteolysis Targeting Chimera) approach for selective ZRANB3 degradation

Researchers exploring therapeutic applications should consider ZRANB3's essentiality in normal tissues and potential redundancy with other DNA repair pathways to develop appropriate targeting strategies.