ZRANB3 Antibody, FITC conjugated

What is ZRANB3 and why is it significant for DNA replication studies?

ZRANB3 (zinc finger, RAN-binding domain-containing protein 3) is a structure-specific ATP-dependent endonuclease that plays a critical role in replication-associated DNA repair . It functions as an SNF2 family ATPase that displays unique structure-specific endonuclease activity, allowing it to cleave branched DNA structures with unusual polarity upon recruitment to stalled replication forks . ZRANB3 is particularly significant because it processes stalled replication forks in association with the replication machinery, providing a novel mechanism for maintaining genomic stability during DNA replication . The study of ZRANB3 is essential for understanding how cells overcome DNA lesions that interfere with processive DNA replication.

What are the key structural domains of ZRANB3 and their functions?

ZRANB3 contains several functional domains that contribute to its role in DNA replication and repair:

• N-terminal helicase core: Characteristic of SNF2 ATPase family members, providing ATP-dependent DNA translocation activity

• PCNA-interacting protein motif (PIP-box): Mediates direct interaction with PCNA, critical for recruitment to replication sites

• NZF-type zinc finger: Binds preferentially to K63-linked polyubiquitin chains, facilitating interaction with ubiquitylated proteins at replication forks

• C-terminal HNH-type endonuclease domain: Confers structure-specific ATP-dependent endonuclease activity

• APIM-motif: Required for interaction with RNR-α, regulating ZRANB3's association with PCNA

These domains work together to facilitate ZRANB3's recruitment to damaged replication forks via multiple mechanisms including interactions with PCNA, K63-polyubiquitin chains, and branched DNA structures .

How does the ZRANB3 Antibody, FITC conjugated differ from non-conjugated antibodies in immunofluorescence applications?

The ZRANB3 Antibody, FITC conjugated offers direct fluorescent detection without requiring secondary antibodies in immunofluorescence applications . This direct detection approach provides several methodological advantages: (1) elimination of potential cross-reactivity associated with secondary antibodies; (2) reduction of background signal in multi-color immunofluorescence experiments; and (3) simplified workflow with fewer incubation and washing steps . When designing co-localization experiments, researchers should consider that the FITC fluorophore (excitation ~495nm, emission ~520nm) emits in the green spectrum, necessitating complementary fluorophores for multi-protein detection . The conjugated antibody allows direct visualization of ZRANB3's localization at replication foci and its recruitment to DNA damage sites, facilitating studies of its spatial relationship with other replication and repair factors.

What are the optimal experimental conditions for detecting ZRANB3 recruitment to stalled replication forks?

For effective detection of ZRANB3 recruitment to stalled replication forks using the FITC-conjugated antibody, implement the following methodology:

• Cell treatment: Induce replication stress with appropriate DNA damaging agents (e.g., mitomycin-C as demonstrated in the literature)

• Fixation protocol: Use 4% paraformaldehyde for 10 minutes at room temperature to preserve protein-DNA interactions

• Permeabilization: Treat with 0.2% Triton X-100 for 5 minutes to allow antibody access

• Blocking: Use 3% BSA in PBS for 30 minutes to reduce non-specific binding

• Antibody dilution: Optimize dilution (typically 1:100 to 1:500) based on signal intensity and background

• Co-staining markers: Include PCNA antibody (different fluorophore) to identify replication foci

• DNA synthesis labeling: Incorporate pulse-labeling with BrdU or EdU to mark active replication sites

• Imaging parameters: Use confocal microscopy with appropriate filter sets for FITC detection

The studies have demonstrated that ZRANB3 localizes to PCNA-containing replication foci and is recruited to these sites following DNA damage . For optimal visualization, capture images of cells in S-phase, identifiable by the characteristic pattern of replication foci.

How should researchers design co-immunoprecipitation experiments to study ZRANB3 interactions with replication machinery?

When designing co-immunoprecipitation (co-IP) experiments to study ZRANB3 interactions with replication machinery components, employ the following methodological approach:

• Lysis conditions: Use mild lysis buffers (e.g., 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, with protease inhibitors) to preserve protein-protein interactions

• Pre-clearing: Incubate lysates with protein G beads for 1 hour to reduce non-specific binding

• Antibody selection: Use anti-ZRANB3 antibody for primary IP to pull down ZRANB3 and its associated proteins

• Controls:

  • Negative control: Use rabbit IgG to assess non-specific binding

  • Input control: Reserve 5-10% of lysate before IP to confirm protein expression

  • PIP-box mutant controls: Include ZRANB3 PIP-box mutants (Q519A, F525A, F526A) to validate PCNA-specific interactions

• Detection strategy: Probe for expected interaction partners (PCNA, MCM proteins) by Western blot

• Reciprocal IP: Perform reverse IP using anti-PCNA antibody to confirm interactions

• Treatment conditions: Compare interactions under normal conditions versus replication stress

Based on published findings, researchers should expect to detect interactions between ZRANB3 and key replication factors including PCNA and several subunits of the MCM helicase complex (MCM3, MCM4, and MCM7) . When implementing PIP-box mutations, the interaction with PCNA should be abrogated while MCM interactions remain intact .

What methodology should be used to investigate the competition between RNR-α and PCNA for ZRANB3 binding?

To investigate the competition between RNR-α and PCNA for ZRANB3 binding, implement this systematic approach:

• In vitro competition assay:

  • Purify recombinant ZRANB3 C-terminal domains (aa 929-1079, containing the HNH-domain and APIM-motif)

  • Purify full-length recombinant RNR-α and PCNA

  • Perform binding experiments with fixed amounts of ZRANB3 and RNR-α, adding increasing concentrations of PCNA

  • Detect complex formation using pull-down assays followed by SDS-PAGE and immunoblotting

• Cellular competition experiments:

  • Overexpress wild-type or reductase-dead (C429S) RNR-α-NLS to increase nuclear RNR-α levels

  • Assess ZRANB3-PCNA interaction by co-IP

  • Compare to control conditions with primarily cytoplasmic RNR-α

• Microscopy-based approach:

  • Induce DNA damage with mitomycin-C

  • Track localization of ZRANB3, RNR-α, and PCNA using fluorescence microscopy

  • Analyze colocalization patterns in small PCNA foci

Based on published findings, expect to observe:

• Reduced ZRANB3-PCNA association when nuclear RNR-α is overexpressed

• Decreased ZRANB3(HNH-APIM)-RNR-α direct interaction in the presence of excess PCNA

• Formation of small PCNA/ZRANB3 foci upon DNA damage that exclude RNR-α

This methodological approach will elucidate the dynamics of the RNR-α-ZRANB3-PCNA axis, wherein nuclear RNR-α levels modulate the degree of ZRANB3 association with PCNA until DNA damage triggers ZRANB3 re-association with PCNA for DNA damage tolerance .

How can the ZRANB3 Antibody, FITC conjugated be utilized to investigate the structure-specific endonuclease activity at replication forks?

To investigate ZRANB3's structure-specific endonuclease activity at replication forks using the FITC-conjugated antibody, implement this advanced methodology:

• Chromatin fractionation with immunofluorescence:

  • Treat cells with replication stress-inducing agents

  • Extract soluble proteins while preserving chromatin-bound factors

  • Perform immunofluorescence with the FITC-conjugated ZRANB3 antibody

  • Co-stain with markers of nascent DNA (EdU) and DNA damage (γH2AX)

  • Quantify ZRANB3 recruitment to damaged replication sites

• In situ proximity ligation assay (PLA):

  • Use ZRANB3 antibody in combination with antibodies against structure-specific DNA intermediates

  • Implement PLA to visualize and quantify direct associations

  • Compare wild-type cells with those expressing nuclease-deficient ZRANB3 mutants

  • Analyze the frequency of PLA signals at replication sites

• DNA fiber analysis with immunodetection:

  • Pulse-label replicating DNA with CldU/IdU

  • Introduce replication stress

  • Combine DNA fiber spreading with immunodetection of ZRANB3-FITC

  • Analyze correlation between ZRANB3 localization and replication fork reversal or restart events

The ZRANB3 antibody enables visualization of enzyme recruitment to specific DNA structures formed during replication stress . Published research demonstrates that ZRANB3 exhibits unusual structure-specific ATP-dependent endonuclease activity, cleaving branched DNA structures with unique polarity to generate accessible 3′-OH groups in the template of the leading strand . This methodology allows researchers to correlate ZRANB3's localization with its enzymatic processing of stalled replication forks in living cells.

What strategies can researchers employ to differentiate between ZRANB3's role in normal replication versus its function in DNA damage response?

To differentiate between ZRANB3's role in normal replication versus DNA damage response, implement this multi-faceted strategy:

• Temporal analysis of ZRANB3 recruitment:

  • Synchronize cells at the G1/S boundary

  • Release into S-phase with and without DNA damaging agents

  • Track ZRANB3 localization using the FITC-conjugated antibody at precisely timed intervals

  • Quantify colocalization with PCNA during unperturbed versus stressed replication

• Domain-specific mutant analysis:

  • Generate cell lines expressing ZRANB3 with specific mutations in:

    • PIP-box (disrupts PCNA interaction)

    • NZF domain (disrupts K63-polyubiquitin binding)

    • HNH domain (disrupts endonuclease activity)

    • APIM motif (disrupts RNR-α interaction)

  • Compare recruitment dynamics and functional outcomes of each mutant

• Replication stress markers correlation:

  • Analyze correlation between ZRANB3 foci and markers of replication stress (RPA foci, γH2AX)

  • Implement machine learning approaches to classify patterns of ZRANB3 distribution in normal versus stressed conditions

How can researchers investigate the functional relationship between ZRANB3's ATP-dependent helicase activity and its endonuclease function?

To investigate the functional relationship between ZRANB3's ATP-dependent helicase activity and its endonuclease function, employ the following advanced methodological approach:

• Structure-function analysis with domain-specific mutations:

  • Generate recombinant ZRANB3 variants with mutations in:

    • Walker A/B motifs (K84A, D157A) to disrupt ATPase activity

    • HNH domain (H1045A) to disrupt endonuclease activity

    • Double mutants affecting both domains

  • Purify these proteins for biochemical assays

• In vitro enzymatic assays:

  • DNA binding assay: Electrophoretic mobility shift assay (EMSA) with different DNA structures

  • ATPase assay: Measure ATP hydrolysis rates with various DNA substrates

  • Helicase assay: Use fluorescently labeled splayed duplexes as described in the literature

  • Endonuclease assay: Employ branched DNA structures to assess cleavage activity

  • Sequential activity assay: Determine whether ATP hydrolysis must precede DNA cleavage

• Cellular complementation studies:

  • Deplete endogenous ZRANB3 using siRNA or CRISPR/Cas9

  • Reconstitute with wild-type or mutant ZRANB3 variants

  • Challenge with replication stress-inducing agents

  • Assess DNA damage markers, replication progression, and genomic stability

Research demonstrates that ZRANB3 catalyzes the time-dependent conversion of splayed DNA duplexes, coupling ATP hydrolysis to its DNA processing activities . The enzyme's unique structure-specific endonuclease activity generates an accessible 3′-OH group in the template of the leading strand, likely facilitating subsequent repair steps . This methodological approach will reveal whether these activities operate independently or are mechanistically coupled, informing models of how ZRANB3 processes stalled replication forks in vivo.

What are the most common technical challenges when using ZRANB3 Antibody, FITC conjugated for immunofluorescence, and how can they be addressed?

When performing immunofluorescence with ZRANB3 Antibody, FITC conjugated, researchers may encounter these common technical challenges:

• High background fluorescence:

  • Cause: Inadequate blocking or excessive antibody concentration

  • Solution: Increase blocking time (1-2 hours), use 5% BSA or 10% normal serum, and titrate antibody concentration (start with 1:200 dilution and adjust as needed)

• Weak signal intensity:

  • Cause: Insufficient antigen retrieval or low ZRANB3 expression

  • Solution: Optimize antigen retrieval methods (heat-induced or enzymatic), increase antibody concentration, or extend incubation time to overnight at 4°C

• Photobleaching of FITC signal:

  • Cause: FITC is relatively susceptible to photobleaching

  • Solution: Add anti-fade reagents to mounting medium, minimize exposure during imaging, capture FITC images first in multi-channel experiments

• Autofluorescence interference:

  • Cause: Cellular components (especially after fixation with aldehydes)

  • Solution: Include a quenching step (0.1-1% sodium borohydride for 5 minutes) after fixation, use Sudan Black B (0.1-0.3%) to reduce autofluorescence

• Nuclear visualization challenges:

  • Cause: ZRANB3's diffuse nuclear pattern outside of replication foci

  • Solution: Use deconvolution microscopy or structured illumination to enhance resolution, counterstain with DAPI to define nuclear boundaries

For optimal results, store the antibody in aliquots at -20°C or -80°C and avoid repeated freeze-thaw cycles as specified in the product information . The antibody is optimally preserved in 50% glycerol with 0.03% Proclin 300 in PBS (pH 7.4) .

How can researchers validate the specificity of ZRANB3 Antibody, FITC conjugated in their experimental system?

To validate the specificity of ZRANB3 Antibody, FITC conjugated in your experimental system, implement this comprehensive validation strategy:

• Genetic validation:

  • Perform ZRANB3 knockdown (siRNA/shRNA) or knockout (CRISPR/Cas9)

  • Compare antibody staining between normal and ZRANB3-depleted cells

  • Expect significant reduction in signal intensity in depleted samples

• Expression validation:

  • Transiently overexpress tagged ZRANB3 (e.g., Flag-tagged)

  • Perform dual-labeling with FITC-conjugated ZRANB3 antibody and anti-tag antibody

  • Analyze colocalization to confirm specificity for ZRANB3

• Western blot validation:

  • Perform western blot with the unconjugated version of the same antibody

  • Verify band at the expected molecular weight (~123 kDa)

  • Include positive control (e.g., cell line known to express ZRANB3) and negative control

• Immunoprecipitation validation:

  • Use the antibody to immunoprecipitate ZRANB3

  • Confirm interaction with known binding partners (PCNA, MCM proteins)

  • Verify that expected mutations (e.g., PIP-box mutations) disrupt specific interactions

• Functional validation:

  • Induce DNA damage and verify recruitment to replication stress sites

  • Confirm colocalization with established replication fork markers

  • Evaluate cell cycle-dependent patterns consistent with ZRANB3 function

The antibody should recognize human ZRANB3 protein as it was raised against recombinant human ZRANB3 protein (amino acids 370-624) . When properly validated, you should observe a pattern of nuclear foci that colocalizes with sites of DNA synthesis (BrdU incorporation) and PCNA, as demonstrated in the research literature .

What considerations should be made when designing quantitative experiments to measure ZRANB3 recruitment dynamics using the FITC-conjugated antibody?

When designing quantitative experiments to measure ZRANB3 recruitment dynamics with FITC-conjugated antibody, consider these methodological parameters:

• Fluorophore stability considerations:

  • FITC is sensitive to photobleaching and pH fluctuations

  • Implement internal fluorescence standards for normalization

  • Consider time-course experiment design with minimal exposures

  • Use identical acquisition parameters across experimental conditions

• Temporal resolution optimization:

  • For live-cell imaging: balance temporal resolution against photobleaching

  • For fixed cells: create precise time-point series after treatment

  • Include synchronization protocols to normalize for cell cycle position

  • Consider automated image acquisition to ensure consistent timing

• Spatial analysis parameters:

  • Define quantification regions precisely (whole nucleus vs. foci)

  • Implement automated foci detection algorithms with consistent thresholds

  • Measure both intensity and morphological parameters (size, number of foci)

  • Consider 3D analysis to capture the complete nuclear distribution

• Experimental controls:

  • Include non-specific IgG-FITC controls to establish background thresholds

  • Use ZRANB3 domain mutants as biological controls (PIP-box, NZF, HNH)

  • Include treatments that alter ZRANB3 dynamics (e.g., nuclear RNR-α overexpression)

  • Implement technical replicate fields and biological replicate experiments

• Quantification methodology:

  • For foci analysis: count number, intensity, and size of foci per nucleus

  • For colocalization: calculate Pearson's or Mander's coefficients with PCNA

  • For recruitment kinetics: measure fluorescence recovery after photobleaching

  • For concentration changes: implement calibration with known standards

Published research demonstrates that ZRANB3 exhibits dynamic recruitment to replication foci that can be modulated by various factors including interactions with PCNA and RNR-α . Statistical analysis should include appropriate tests for the distribution of your data (parametric or non-parametric) with correction for multiple comparisons when analyzing across different conditions or time points.

How should researchers interpret changes in ZRANB3 localization patterns following different types of replication stress?

When interpreting changes in ZRANB3 localization patterns following different types of replication stress, consider these analytical frameworks:

• Pattern classification based on stressor type:

  • Interstrand crosslinks (e.g., mitomycin-C): Expect discrete ZRANB3 foci that colocalize with PCNA but exclude RNR-α

  • Alkylating agents (e.g., MMS): Anticipate broader ZRANB3 recruitment to damaged regions

  • Replication inhibitors (e.g., aphidicolin): Look for accumulation at stalled fork boundaries

  • Topoisomerase inhibitors: Analyze association with DNA-protein crosslinks

• Temporal dynamics interpretation:

  • Early response (minutes): Initial recruitment via PCNA interaction (PIP-box dependent)

  • Intermediate response (30-120 minutes): Stabilization through K63-polyubiquitin binding (NZF-dependent)

  • Late response (2-24 hours): Association with persistent damage or repair intermediates

• Colocalization patterns analysis:

  • ZRANB3+PCNA+RPA: Indicates exposure of ssDNA at stalled forks

  • ZRANB3+PCNA+γH2AX: Suggests processing of damaged replication forks

  • ZRANB3+PCNA−RNR-α: Consistent with the competition model between RNR-α and PCNA

• Statistical interpretation guidelines:

  • Quantify percentage of cells showing different ZRANB3 patterns

  • Analyze average number and intensity of foci per nucleus

  • Compare kinetics of formation/resolution across different stressors

  • Correlate patterns with cell cycle markers and replication status

Research evidence demonstrates that ZRANB3 is recruited to DNA breaks and stressed replication forks, and its deficiency leads to increased susceptibility to DNA damage induced by agents like MMS . The recruitment involves multiple mechanisms, leveraging ZRANB3's structural domains for interactions with PCNA, K63-polyubiquitin chains, and branched DNA structures . When properly analyzed, changes in ZRANB3 localization can provide valuable insights into the cellular response to different types of replication stress.

What are the implications of the competition between RNR-α and PCNA for ZRANB3 binding in understanding DNA replication and repair mechanisms?

The competition between RNR-α and PCNA for ZRANB3 binding has several significant implications for understanding DNA replication and repair mechanisms:

• Regulatory mechanism for ZRANB3 activity:

  • Nuclear RNR-α forms a complex with ZRANB3 via the APIM-motif, preventing ZRANB3-PCNA interaction in undamaged cells

  • This competition creates a molecular switch that controls ZRANB3's participation in normal replication versus damage response

  • Nuclear levels of RNR-α may therefore serve as a modulator of ZRANB3's association with the replication machinery

• Cell cycle control implications:

  • The balance between RNR-α-bound and PCNA-bound ZRANB3 likely shifts during cell cycle progression

  • This may coordinate ZRANB3's endonuclease activity with the cell's replicative status

  • Changes in nuclear RNR-α levels could regulate ZRANB3-dependent fork processing throughout S-phase

• Damage response pathway integration:

  • Upon DNA damage, ZRANB3 re-associates with PCNA at damage sites, overriding the RNR-α interaction

  • This suggests a hierarchical organization of ZRANB3's binding preferences dependent on cellular stress

  • The competition may serve as a checkpoint preventing inappropriate nucleolytic processing during normal replication

• Therapeutic targeting potential:

  • Modulating the RNR-α-ZRANB3-PCNA axis could provide novel approaches for enhancing DNA damage sensitivity

  • Small molecules disrupting specific interactions could selectively affect either normal replication or damage response

  • Understanding this competition offers new targets for combination therapies in DNA repair-deficient cancers

Research demonstrates that overexpression of either reductase-dead(C429S) or wild-type RNR-α-NLS reduces ZRANB3-PCNA association, while excess PCNA reduces ZRANB3(HNH-APIM)-RNR-α direct interaction . This competition model provides a mechanistic explanation for how cells ensure appropriate deployment of ZRANB3's endonuclease activity only when needed for DNA damage tolerance.

How does the structure-specific endonuclease activity of ZRANB3 contribute to replication fork stability, and what are the implications for genomic integrity?

The structure-specific endonuclease activity of ZRANB3 contributes to replication fork stability through several mechanistic pathways with significant implications for genomic integrity:

• Processing of specialized DNA structures:

  • ZRANB3 cleaves branched DNA structures with unusual polarity, generating accessible 3′-OH groups in the template of the leading strand

  • This activity allows resolution of complex DNA structures that arise during replication stress

  • The ATP-dependent nature of this activity couples energy expenditure to appropriate fork processing

• Replication fork remodeling functions:

  • ZRANB3's endonuclease activity may facilitate fork reversal, a protective mechanism during replication stress

  • Through controlled incision, ZRANB3 could promote template switching to bypass DNA lesions

  • The generated 3′-OH ends serve as primers for DNA synthesis during replication restart

• Integration with DNA damage tolerance pathways:

  • ZRANB3 recruitment via PCNA and K63-polyubiquitin chains links its activity to the DNA damage tolerance network

  • This integration ensures that structure-specific cleavage occurs in the context of appropriate damage signaling

  • The interaction with PCNA positions ZRANB3 for action specifically at replication-associated damage sites

• Genomic integrity implications:

  • Proper ZRANB3 function prevents replication fork collapse and double-strand break formation

  • Deficiency leads to increased susceptibility to DNA damaging agents like MMS

  • Unregulated ZRANB3 activity could potentially lead to inappropriate DNA cleavage and genomic instability

The regulation of ZRANB3's endonuclease activity via multiple mechanisms, including competition with RNR-α and ATP-dependency , highlights the critical nature of this enzyme in maintaining genomic stability. The generation of specific DNA ends by ZRANB3 endonuclease activity likely directs subsequent repair pathway choice, influencing how cells respond to and recover from replication stress.