ZRANB3 Antibody, Biotin conjugated

What is ZRANB3 and what is its role in DNA replication and repair?

ZRANB3 is a SNF2 family ATPase that plays a specialized role in replication-associated DNA repair. It possesses a unique structure-specific endonuclease activity contained within its C-terminal HNH domain that allows it to cleave branched DNA structures with unusual polarity, generating an accessible 3′-OH group in the template of the leading strand . ZRANB3 localizes to DNA replication sites and interacts with key replication factors including PCNA and subunits of the replicative helicase MCM complex (MCM3, MCM4, and MCM7) . Upon DNA damage, ZRANB3 is rapidly recruited to stressed replication forks through multiple mechanisms involving interactions with PCNA, K63-polyubiquitin chains, and branched DNA structures . Functionally, ZRANB3 deficiency leads to increased susceptibility to DNA damage induced by methylmethane sulfonate (MMS), highlighting its importance in maintaining genomic stability during replication .

How does a biotin-conjugated ZRANB3 antibody differ from unconjugated versions in experimental applications?

Biotin-conjugated ZRANB3 antibodies offer several methodological advantages while maintaining the specificity of unconjugated versions. The biotin conjugation enables signal amplification through the high-affinity biotin-streptavidin interaction, which can significantly enhance detection sensitivity in various applications. This feature is particularly valuable when studying proteins like ZRANB3 that may be present at relatively low abundance at replication sites or DNA damage foci.

In proximity ligation assays (PLA), biotin-conjugated antibodies can be effectively paired with streptavidin-linked oligonucleotides to detect protein-protein interactions or protein-DNA associations . For example, researchers have successfully used biotin conjugation in combination with EdU labeling and PLA to visualize ZRANB3 recruitment to nascent DNA at replication forks . The biotin-conjugated format also facilitates multiplexing in immunofluorescence experiments, allowing simultaneous detection of ZRANB3 alongside other replication factors using orthogonal detection systems.

When selecting between conjugated and unconjugated antibodies, researchers should consider that while the biotin molecule is relatively small, the conjugation process may occasionally affect antibody binding characteristics, particularly if conjugation occurs near the epitope recognition site.

What cellular localization pattern should I expect when using a ZRANB3 antibody?

ZRANB3 displays distinct nuclear localization patterns that vary depending on cell cycle phase and DNA damage status. Under normal conditions, ZRANB3 shows a patterned nuclear distribution reminiscent of replication foci . These foci colocalize with sites of active DNA synthesis as demonstrated by their overlap with bromodeoxyuridine (BrdU) incorporation and PCNA staining .

Upon induction of DNA damage by agents like MMS or laser microirradiation, ZRANB3 rapidly relocates to sites of DNA damage within the first minute following damage induction . The kinetics of this recruitment are comparable to replication factors like PCNA and FEN1 but slower than chromatin remodelers like ALC1 . Unlike ALC1 recruitment, ZRANB3 localization to damage sites is not inhibited by PARP inhibitors, indicating a PARP-independent recruitment mechanism .

In experimental settings, fluorescently tagged ZRANB3 (e.g., YFP-ZRANB3) typically appears as distinct nuclear foci in S-phase cells, with more diffuse nuclear staining in other cell cycle phases. When using immunofluorescence with biotin-conjugated ZRANB3 antibodies, optimize fixation and permeabilization conditions to preserve nuclear architecture and ensure antibody accessibility to nuclear replication compartments.

Which validated cell lines are recommended for studying ZRANB3 function?

Based on published research, several cell lines have been validated for ZRANB3 studies:

When selecting a cell line for your experiments, consider factors such as endogenous ZRANB3 expression levels, efficiency of transfection/transduction for exogenous expression, and relevant biological context for your research question. Western blot analysis using validated ZRANB3 antibodies shows detectable expression across multiple cell lines, though expression levels may vary .

What controls should be included when using ZRANB3 antibodies in Western blotting?

When performing Western blot analysis with biotin-conjugated ZRANB3 antibodies, include the following essential controls:

Positive Controls:

• Lysates from cells overexpressing ZRANB3 (tagged or untagged)

• Well-characterized cell lines known to express ZRANB3 (U2OS, 293T)

• Recombinant ZRANB3 protein (full-length or fragment containing the antibody epitope)

Negative Controls:

• ZRANB3 knockdown or knockout cell lysates

• Cell lines with naturally low ZRANB3 expression

• Blocking with immunizing peptide (if available)

Technical Controls:

• Streptavidin-only control (without primary antibody) to assess non-specific binding

• Loading controls appropriate for nuclear proteins (Lamin B1, Histone H3)

• Molecular weight markers to confirm detection at the expected size (~123 kDa)

Validation Approach:

• Test antibody at multiple dilutions (1:1000-1:3000 recommended for Western blotting)

• Confirm detection of a single band at approximately 123 kDa

• Verify signal reduction in ZRANB3-depleted samples

• Include positive controls from different cell types to assess expression variation

For enhanced specificity when using biotin-conjugated antibodies, consider pre-clearing lysates with streptavidin beads before immunoblotting to reduce background, and use biotin-blocking reagents to minimize non-specific streptavidin binding during detection.

How can I optimize detection of ZRANB3 recruitment to stalled replication forks?

Detecting ZRANB3 recruitment to stalled replication forks requires optimized protocols that capture this dynamic process. Consider these methodological approaches:

Fluorescence Microscopy Optimization:

• Synchronize cells in early S-phase using double thymidine block or hydroxyurea pre-treatment

• Induce fork stalling with low-dose aphidicolin (0.2-0.5 μM) or hydroxyurea (2-4 mM)

• For optimal fixation, use 4% paraformaldehyde with 0.1% Triton X-100 permeabilization

• Enhance detection sensitivity using tyramide signal amplification with biotin-conjugated antibodies

• Co-stain with replication fork markers (PCNA, EdU incorporation) to confirm localization

Biochemical Approaches:

• Use iPOND (isolation of Proteins On Nascent DNA) to capture proteins at replication forks:

  • Pulse-label replicating DNA with EdU

  • Conjugate biotin to EdU by click chemistry

  • Perform streptavidin pulldown to isolate replication fork proteins

  • Detect ZRANB3 by Western blotting

• Perform proximity ligation assays (PLA) to detect ZRANB3 at specific fork structures:

  • Use biotin-conjugated ZRANB3 antibody paired with antibodies against fork components

  • Alternatively, use anti-HA antibodies with biotin-labeled nascent DNA

Enhanced Detection Strategies:

• Deplete the PCNA deubiquitinase USP1 to increase PCNA ubiquitination and enhance ZRANB3 recruitment

• Ensure RFWD3 function is intact, as it promotes ZRANB3 recruitment to stalled forks

• Consider using ZRANB3 constructs with mutations in the PIP-box, APIM, or NZF domains as controls to validate recruitment mechanisms

• Implement super-resolution microscopy (STORM, SIM) to visualize ZRANB3 localization at individual replication forks

This multi-faceted approach will optimize detection of ZRANB3 at replication forks and facilitate analysis of its recruitment dynamics under various experimental conditions.

What experimental approaches can distinguish between ZRANB3's ATPase and endonuclease activities?

ZRANB3 possesses both ATPase and endonuclease activities that are functionally coupled but mechanistically distinct. To dissect these activities:

Domain-Specific Mutational Analysis:

Biochemical Activity Assays:

• ATPase activity:

  • Measure ATP hydrolysis using malachite green phosphate detection

  • Test activity on different DNA substrates (ssDNA, dsDNA, branched structures)

  • Analyze enzyme kinetics (Km, Vmax) with varying substrate concentrations

• Endonuclease activity:

  • Design fluorescently labeled branched DNA substrates

  • Analyze cleavage products by denaturing gel electrophoresis

  • Map precise cleavage sites by sequencing or primer extension

• Coupled assay development:

  • Monitor ATP hydrolysis and DNA cleavage simultaneously

  • Determine temporal relationship between activities

  • Test effect of ATP analogs (ATPγS, AMP-PNP) on nuclease function

Advanced Structural Approaches:

• Hydrogen-deuterium exchange mass spectrometry to identify conformational changes upon ATP binding

• Single-molecule FRET to detect structural transitions during catalytic cycle

• Cryo-EM analysis of ZRANB3 bound to branched DNA substrates with/without ATP

These approaches will help delineate the mechanistic relationship between ZRANB3's ATPase and endonuclease activities, clarifying how ATP hydrolysis might drive conformational changes that enable structure-specific DNA cleavage with the unusual polarity observed in biochemical studies .

How does RFWD3 influence ZRANB3 recruitment and function at replication forks?

The ubiquitin ligase RFWD3 plays a crucial role in regulating ZRANB3 recruitment to stalled replication forks through several mechanisms:

RFWD3-Dependent ZRANB3 Recruitment:
RFWD3 promotes ZRANB3 recruitment to stalled replication forks in a manner that depends on its ubiquitin ligase activity . This recruitment pathway is functionally significant, as demonstrated by electron microscopy studies showing that RFWD3 stimulates fork remodeling in a ZRANB3-epistatic manner . When RFWD3 is depleted or inactivated, ZRANB3 localization to replication forks is impaired, particularly following replication stress .

Mechanistic Basis:
RFWD3 likely promotes ZRANB3 recruitment by enhancing PCNA polyubiquitination. While monoubiquitination of PCNA is primarily mediated by RAD18, polyubiquitin chain extension involves multiple E3 ligases including HLTF, SHPRH, and RFWD3 . The NZF domain of ZRANB3 specifically recognizes K63-linked polyubiquitin chains , providing a direct mechanistic link between RFWD3-mediated ubiquitination and ZRANB3 recruitment.

Functional Significance in Fork Protection:
The RFWD3-ZRANB3 pathway plays a critical role in fork remodeling, which becomes particularly evident in BRCA2-deficient backgrounds. RFWD3 depletion in BRCA2-deficient cells rescues nascent DNA degradation and fork collapse into double-strand breaks, phenocopying the effects of ZRANB3 inactivation . This indicates that RFWD3 and ZRANB3 function in the same pathway to promote fork reversal.

Experimental Approaches to Study This Relationship:

• Compare ZRANB3 recruitment in RFWD3-proficient versus RFWD3-depleted cells using fluorescence microscopy

• Analyze PCNA ubiquitination status in parallel with ZRANB3 recruitment

• Perform epistasis analysis by depleting both proteins individually and in combination

• Use catalytically inactive RFWD3 mutants to confirm the requirement for its ubiquitin ligase activity

• Analyze fork structures by electron microscopy in cells with different RFWD3/ZRANB3 status

This RFWD3-ZRANB3 regulatory axis represents an important mechanism for controlling replication fork remodeling in response to genotoxic stress, with implications for genome stability and cancer development.

What methodological approaches can resolve contradictory findings regarding ZRANB3 function across different experimental systems?

Reconciling contradictory results regarding ZRANB3 function requires systematic methodological approaches:

Standardized Experimental Systems:

• Create a panel of isogenic cell lines with ZRANB3 knockout/knockdown using identical methodology

• Develop reconstitution systems with wild-type and mutant ZRANB3 at physiological expression levels

• Establish standard protocols for key assays (fork reversal measurement, replication stress response, DNA damage sensitivity)

Comprehensive Functional Analysis:

Factors Contributing to Experimental Variation:

• Cell cycle distribution differences between experimental systems

• Varying expression levels of ZRANB3 interaction partners (PCNA, RFWD3)

• Differences in replication stress induction methods

• Variations in PCNA ubiquitination status across cell types

• Compensatory mechanisms involving alternative fork remodelers

Reconciliation Strategy:

• Directly compare ZRANB3 recruitment kinetics across cell lines using identical imaging conditions

• Profile the expression of known ZRANB3 interactors in different experimental systems

• Analyze post-translational modifications of ZRANB3 that might affect its function

• Perform careful epistasis analysis with other replication stress response factors

• Develop computational models that integrate multiple datasets to identify context-dependent functions

By implementing these approaches, researchers can identify the cellular contexts and molecular conditions in which specific ZRANB3 functions predominate, reconciling seemingly contradictory findings and developing a more nuanced understanding of its role in genome maintenance.

How can I design experiments to study the interaction between ZRANB3 and ubiquitinated PCNA?

The interaction between ZRANB3 and ubiquitinated PCNA is central to ZRANB3's recruitment to stalled replication forks. Design robust experiments using these approaches:

Biochemical Interaction Studies:

• In vitro reconstitution system:

  • Purify recombinant ZRANB3 (full-length and domain mutants)

  • Generate mono- and polyubiquitinated PCNA using purified E1, E2, and E3 enzymes

  • Perform binding assays with increasing stringency to determine specificity and affinity

  • Use surface plasmon resonance or microscale thermophoresis to measure binding kinetics

• Domain mapping experiments:

  • Create GST-fusion constructs of ZRANB3 domains (NZF, PIP-box, APIM)

  • Test binding to different forms of ubiquitinated PCNA

  • Analyze the contribution of K63 versus K48 polyubiquitin chains

  • Perform competition assays with ubiquitin-binding domains from other proteins

Cellular Visualization Methods:

• Proximity ligation assay (PLA):

  • Use biotin-conjugated ZRANB3 antibody with anti-PCNA antibody

  • Include antibodies specific for ubiquitinated PCNA

  • Quantify PLA signals under normal conditions versus replication stress

  • Compare wild-type cells with those expressing ZRANB3 domain mutants

• Fluorescence complementation:

  • Split fluorescent protein between ZRANB3 and PCNA

  • Monitor fluorescence reconstitution under various conditions

  • Test the effect of ubiquitination inhibitors or deubiquitinase overexpression

Genetic Manipulation Approaches:

• PCNA mutant analysis:

  • Express PCNA-K164R mutant (cannot be ubiquitinated)

  • Test ZRANB3 recruitment in response to replication stress

  • Compare with other PCNA mutants that affect protein interactions

• E3 ligase manipulation:

  • Deplete or inhibit specific E3 ligases (RAD18, HLTF, SHPRH, RFWD3)

  • Analyze changes in ZRANB3-PCNA interaction

  • Perform rescue experiments with wild-type versus catalytically inactive E3 ligases

• USP1 depletion:

  • Enhance PCNA ubiquitination by depleting the deubiquitinase USP1

  • Quantify ZRANB3 recruitment to spontaneous nuclear foci

  • Compare recruitment of wild-type ZRANB3 versus NZF domain mutants

Data Analysis and Integration:

• Correlate PCNA ubiquitination levels with ZRANB3 recruitment efficiency

• Develop quantitative models of the relationship between ubiquitination states and ZRANB3 binding

• Integrate structural information about the ZRANB3 NZF domain to interpret interaction data

These experimental approaches will provide comprehensive insights into the molecular mechanisms governing ZRANB3 recognition of ubiquitinated PCNA and help resolve important questions about specificity, regulation, and functional outcomes of this interaction.

What techniques can measure ZRANB3's enzymatic activities in cellular extracts?

Assessing ZRANB3's enzymatic activities in cellular contexts requires specialized techniques that preserve its native state and interactions:

ATPase Activity Measurements:

• Immunoprecipitation-coupled ATPase assay:

  • Immunoprecipitate ZRANB3 from cell extracts using biotin-conjugated antibodies

  • Perform on-bead ATPase assays using radiolabeled ATP (γ-³²P-ATP)

  • Measure released phosphate by thin-layer chromatography or malachite green assay

  • Include negative controls (ATP-binding mutant K65R, mock immunoprecipitation)

• ZRANB3 activity in cellular fractions:

  • Fractionate cells into cytoplasmic, nucleoplasmic, and chromatin-bound fractions

  • Measure ATPase activity in each fraction with/without added DNA substrates

  • Correlate activity with ZRANB3 protein levels by Western blotting

  • Compare activity before and after replication stress induction

Nuclease Activity Detection:

• Immunoprecipitation-coupled nuclease assay:

  • Immunoprecipitate ZRANB3 from cells

  • Incubate with fluorescently labeled branched DNA substrates

  • Analyze cleavage products by denaturing PAGE

  • Compare activities before/after replication stress induction

• In-gel nuclease activity assay:

  • Separate cellular proteins on native PAGE containing branched DNA substrates

  • Incubate gel in reaction buffer containing ATP and divalent cations

  • Visualize regions of DNA cleavage under UV illumination

  • Confirm ZRANB3 responsibility by Western blotting or mass spectrometry

Combined Activity Approaches:

Activation State Analysis:

• Phospho-specific antibodies to detect potential regulatory modifications

• Chemical crosslinking to capture ZRANB3 interaction partners that might modulate activity

• ATP-binding assays using fluorescent ATP analogs to assess nucleotide-binding capacity

These methodologies will enable researchers to monitor ZRANB3's enzymatic activities in physiologically relevant contexts, providing insights into how its functions are regulated in response to replication stress and DNA damage.