POLE3 Antibody

What is POLE3 and why is it significant in molecular biology research?

POLE3 (Polymerase Epsilon Subunit 3) is a non-essential 17 kDa subunit of the DNA polymerase epsilon (POLE) holoenzyme that functions as a histone H3-H4 chaperone maintaining chromatin integrity during DNA replication . Recent research has revealed POLE3's significance in:

• Transcriptional repression of unintegrated HIV-1 DNA

• Maintaining chromatin structure during replication

• Contributing to genome stability

• Potential therapeutic applications in cancer treatment

The protein's dual role in DNA replication and chromatin maintenance makes it an important target for researchers studying fundamental DNA processes and disease mechanisms .

What experimental applications are validated for commercial POLE3 antibodies?

Current commercial POLE3 antibodies have been validated for multiple applications with specific performance characteristics:

For optimal results, researchers should perform antibody titration for their specific experimental system .

How should researchers optimize POLE3 antibody protocols for different cell types?

Optimization strategies depend on the cell type and application:

For Western blot:

• Begin with recommended dilutions (1:1000-1:4000)

• For difficult-to-detect samples, increase antibody concentration and optimize lysis conditions

• HeLa, MCF-7, and HepG2 cells have confirmed positive detection

For immunohistochemistry:

• Test both TE buffer pH 9.0 and citrate buffer pH 6.0 for antigen retrieval

• Human liver cancer tissue shows optimal detection

• For formalin-fixed tissues, extend antigen retrieval time

For immunofluorescence:

• Begin with 1:100 dilution for adherent cells

• Optimize fixation method (4% paraformaldehyde versus methanol)

• Permeabilization conditions may require adjustment based on subcellular localization

How can POLE3 antibodies be utilized to study HIV-1 silencing mechanisms?

Recent research has established POLE3 as a transcriptional repressor of unintegrated HIV-1 DNA. Researchers can leverage POLE3 antibodies to:

• Perform chromatin immunoprecipitation (ChIP) or CUT&RUN assays to analyze POLE3 loading onto unintegrated HIV-1 DNA

• Use POLE3 knockdown followed by antibody-based detection to examine changes in:

  • H3 loading (remains unchanged)

  • Active histone mark H3Ac (increases 2.1-fold)

  • RNAPII recruitment (increases significantly)

  • H1.2 loading (shows small but significant reduction)

Methodology: Use cleavage under targets and release using nuclease (CUT&RUN) with POLE3 antibodies to investigate POLE3 accumulation on unintegrated HIV-1 DNA. Specific viral DNA sequences can be recovered from POLE3 immunoprecipitation using chromatin from control cells .

What mechanisms explain the relationship between POLE3-POLE4 and PARP inhibitor sensitivity?

Research shows that loss of POLE3-POLE4 sensitizes cells to PARP inhibitors (PARPi) through mechanisms parallel to BRCA1 deficiency:

• POLE4 loss affects replication speed rather than DNA damage response

• This leads to:

  • Accumulation of single-stranded DNA gaps behind replication forks upon PARPi treatment

  • Impaired post-replicative repair

  • Elevated replication stress signaling involving ATR and DNA-PK

Notably, POLE4 acts parallel to BRCA1 in inducing PARPi sensitivity and may counteract acquired resistance associated with homologous recombination restoration .

For experimental investigation, researchers can use POLE3 antibodies to:

• Confirm knockout/knockdown efficiency

• Examine protein interactions during replication stress

• Analyze chromatin association during PARPi treatment

How can researchers distinguish between POLE3's DNA polymerase and histone chaperone functions using antibody-based techniques?

POLE3's dual functionality requires specific experimental approaches to distinguish between its roles:

Polymerase function analysis:

• Perform immunoprecipitation with POLE3 antibodies followed by interaction studies with POLE catalytic subunits

• Use proximity ligation assays (PLA) to detect POLE3 association with replication factors

Histone chaperone function analysis:

• Use co-immunoprecipitation with POLE3 antibodies to detect H3-H4 binding

• Apply hydrogen/deuterium exchange mass spectrometry after POLE3 immunoprecipitation

• Western blotting can confirm POLE3-POLE4 binding to both H3-H4 dimers and tetramers at physiological salt concentrations (150 mM NaCl) and at higher concentrations (300 mM NaCl)

Experimental studies have confirmed that POLE3-POLE4 can bind to H3-H4 dimers containing H3 mutations (H3 C110E) that prevent H3-H4 tetramerization, supporting its function as a bona fide histone chaperone .

What are common pitfalls in POLE3 antibody experiments and how can they be addressed?

For cellular applications, researchers should note that POLE3 expression can vary significantly between cell types, with documented detection in HeLa, MCF-7, and HepG2 cells .

How can researchers validate POLE3 antibody specificity for chromatin-associated studies?

For chromatin association studies, specificity validation is critical:

• Knockout/knockdown controls:

  • Include POLE3 knockout/knockdown samples as negative controls

  • Confirm reduced signal in POLE3 ChIP/CUT&RUN experiments from POLE3-depleted cells

• Cross-validation approaches:

  • Use multiple antibodies targeting different POLE3 epitopes

  • Compare monoclonal (e.g., PCRP-POLE3-4E1) and polyclonal (e.g., 15278-1-AP) antibodies

  • Validate with tagged POLE3 constructs (FLAG/HA) using tag-specific antibodies

• Competitive peptide blocking:

  • Pre-incubate antibody with immunizing peptide before application

  • Confirm signal reduction in Western blot or immunostaining

Research has demonstrated the ability to recover viral DNA sequences in POLE3 immunoprecipitates using chromatin from control cells, with significantly reduced recovery when using chromatin from POLE3-depleted cells, confirming antibody specificity .

What approaches can resolve contradictory results between different POLE3 antibodies?

When facing contradictory results:

• Epitope mapping analysis:

  • Determine if antibodies recognize different domains of POLE3

  • Check if post-translational modifications affect epitope accessibility

  • Consider potential protein isoforms or truncations

• Validation in multiple systems:

  • Test antibodies in various cell types (HeLa, SupT1, RPE-1)

  • Compare results in different experimental contexts (basal vs. stress conditions)

  • Include appropriate positive controls (e.g., cells with known POLE3 expression)

• Advanced validation techniques:

  • Mass spectrometry analysis of immunoprecipitated material

  • Orthogonal detection methods (RNA-seq correlation with protein levels)

  • Proximity labeling approaches to confirm interaction networks

Studies have shown that POLE3's repressive activity toward unintegrated HIV-1 DNA was consistently observed across different cell types including SupT1 cell lines and hTERT-immortalized RPE-1 human retinal pigment epithelial cells, demonstrating that POLE3 function is not cell-type specific .

How can POLE3 antibodies contribute to understanding HIV-1 infection mechanisms?

POLE3 antibodies enable several novel research approaches for HIV-1 studies:

• Infection stage-specific chromatin dynamics:

  • ChIP-seq or CUT&RUN with POLE3 antibodies at different stages of HIV-1 infection

  • Analysis of POLE3 recruitment kinetics to unintegrated viral DNA

  • Comparative analysis between integration-competent and integration-defective viruses

• Mechanistic studies of HIV-1 silencing:

  • POLE3 knockdown followed by chromatin immunoprecipitation shows:

    • No effect on H3 loading

    • 2.1-fold increase in active histone mark H3Ac

    • Significant increase in RNAPII recruitment

    • Small but significant reduction in H1.2 loading at HIV-1 regulatory regions

• VPR antagonism studies:

  • POLE3's transcriptional repression of unintegrated HIV-1 DNA operates independently of the viral protein R (VPR)

  • Both VPR-positive and VPR-negative viruses show increased transcription upon POLE3 knockdown

This research direction could identify novel therapeutic targets for modulating HIV-1 latency.

What role could POLE3 play in cancer therapy, and how can antibodies facilitate this research?

POLE3's emerging role in cancer therapy, particularly related to PARP inhibitors, opens several research avenues:

• PARP inhibitor sensitivity biomarkers:

  • Use POLE3 antibodies for immunohistochemical analysis of tumor samples

  • Correlate POLE3/POLE4 expression with PARPi treatment outcomes

  • Develop predictive assays for patient stratification

• Mechanistic studies of replication stress:

  • POLE3/POLE4 loss affects replication speed and leads to single-stranded DNA gap accumulation

  • This occurs through pathways parallel to BRCA1 deficiency

  • The mechanism involves impaired post-replicative repair and elevated replication stress signaling

• Therapeutic targeting approaches:

  • Develop inhibitors of POLE3-POLE4 interaction

  • Screen for compounds that disrupt POLE3's histone chaperone function

  • Combine POLE3/POLE4 targeting with existing PARP inhibitor therapies

Research shows POLE4 acts parallel to BRCA1 in inducing PARPi sensitivity and may counteract acquired resistance, establishing POLE4 as a promising target to improve PARPi-driven therapies and hamper acquired resistance .

How can POLE3 antibodies illuminate the relationship between DNA replication and chromatin maintenance?

POLE3's dual role presents unique opportunities to study the replication-chromatin interface:

• Chromatin assembly during replication:

  • POLE3-POLE4 functions as a histone H3-H4 chaperone

  • The complex can bind to both H3-H4 dimers and tetramers at physiological salt concentrations

  • It can assemble histones H3-H4 onto linear DNA and relax circular plasmid DNA

• Temporal dynamics of histone deposition:

  • Use synchronized cell populations and POLE3 ChIP-seq

  • Map POLE3 localization relative to replication fork progression

  • Analyze co-localization with newly synthesized versus parental histones

• Structural studies of POLE3-histone interactions:

  • Hydrogen/deuterium exchange mass spectrometry has defined minimal domains necessary for interaction between POLE3-POLE4 and histones H3-H4

  • Physical mapping experiments have characterized the binding interface

  • These approaches have established POLE3-POLE4 as a histone chaperone that promotes tetrasome formation and DNA supercoiling

This research area connects fundamental processes of genome maintenance and offers insights into epigenetic inheritance mechanisms.

What are the optimal fixation and permeabilization conditions for POLE3 immunofluorescence in different subcellular compartments?

Optimization parameters for subcellular localization studies:

For immunofluorescence studies, antibody dilutions of 1:50-1:500 are recommended, though specific optimization for each cell type is advised . HepG2 cells have confirmed positive detection for IF/ICC applications.

How should researchers design ChIP-seq experiments to study POLE3 binding to HIV-1 DNA versus host genomic DNA?

Experimental design recommendations for differential binding studies:

• Sample preparation:

  • Include appropriate controls (uninfected cells, POLE3 knockdown/knockout)

  • Time-course analysis (e.g., 9 hours and 48 hours post-infection)

  • Consider both integration-competent and integration-defective viral variants

• Immunoprecipitation optimization:

  • Cross-linking: 1% formaldehyde, 10 minutes at room temperature

  • Sonication: Adjust to generate 200-500 bp fragments

  • IP conditions: 1-5 μg antibody per ChIP reaction

• Analysis considerations:

  • For viral DNA: Design primers specific to viral LTR regions

  • For host DNA: Genome-wide analysis with peak calling

  • Quantification: qPCR for targeted regions, sequencing for genome-wide analysis

Recent research successfully used cleavage under targets and release using nuclease (CUT&RUN) and ChIP to detect POLE3 and POLE4 accumulation on unintegrated HIV-1 DNA, demonstrating these techniques' viability .

What experimental designs can differentiate between POLE3's direct effects on chromatin versus indirect effects through protein-protein interactions?

To distinguish direct chromatin effects from indirect interactions:

• Domain mutant analysis:

  • Use POLE3 ΔC mutant (lacks histone H3 and H4 chaperone activity)

  • Use POLE3 F44D mutant (cannot interact with POLE4)

  • Compare effects on transcriptional repression and chromatin structure

• Sequential ChIP (Re-ChIP):

  • First IP with POLE3 antibody

  • Second IP with antibodies against histone marks or other factors

  • Identifies chromatin regions where POLE3 co-occurs with specific marks

• Proximity-dependent labeling:

  • APEX2 or BioID fusions to POLE3

  • Identify proteins in close proximity in living cells

  • Distinguish direct interactors from proteins that co-occur on chromatin