Phospho-RPA2 (T21) Recombinant Monoclonal Antibody

What is the biological function of RPA2 and its phosphorylated form at T21?

RPA2 (also known as RPA32) is a 32 kDa subunit of the heterotrimeric Replication Protein A complex (RPA/RP-A) that plays essential roles in DNA metabolism. As part of this complex, RPA2 binds and stabilizes single-stranded DNA intermediates that form during DNA replication or upon DNA stress . The phosphorylation of RPA2 at threonine 21 (T21) occurs primarily during DNA damage responses and is mediated by phosphatidylinositol 3-kinase-related kinases (PIKKs) such as ATR and DNA-PK.

T21 phosphorylation represents a critical threshold in the DNA damage response pathway. Research indicates that Thr21 is the last site to be phosphorylated in the hierarchical phosphorylation cascade of RPA2 and indicates impending replication catastrophe and irreparable damage . This phosphorylation event helps transition RPA from its replication role to its function in DNA repair mechanisms.

How does phosphorylation at T21 affect RPA2 function in DNA damage response?

T21 phosphorylation significantly alters RPA2's functionality in DNA damage response pathways. When phosphorylated at T21, RPA2 facilitates adaptation of DNA-replication forks to replication stress . This modification enhances the recruitment of DNA repair factors and helps coordinate checkpoint responses.

Studies have shown that cells expressing RPA2 with mutations at phosphorylation sites (T21A/S33A) demonstrate:

• Significantly reduced cell proliferation compared to wild-type RPA2 expressing cells

• Only 30% survival under replication stress conditions (5 mM hydroxyurea for 48 hours) versus minimal changes in wild-type RPA2 cells

• Increased apoptosis (~27% of cells become TUNEL-positive after hydroxyurea treatment compared to ~6% in wild-type)

These findings demonstrate that proper RPA2 phosphorylation at T21 is critical for cell viability under both normal and replication stress conditions.

What are the optimal applications for Phospho-RPA2 (T21) antibodies in experimental settings?

Phospho-RPA2 (T21) antibodies have several validated applications across various experimental platforms:

• Western Blotting: Detects a specific band at approximately 32-40 kDa under reducing conditions. Treatment of samples with DNA-damaging agents like camptothecin (1 μM for 1 hour) significantly enhances signal detection .

• Immunofluorescence: Enables visualization and quantification of pRPA2 foci in both cell lines and tissue samples. This approach allows for scoring cells based on foci number (typically cells with ≥2 pRPA2 foci) .

• ELISA/Dot Blot Analysis: Provides high sensitivity and specificity for distinguishing between phosphorylated and non-phosphorylated forms, as demonstrated by direct ELISA antigen dose-response curves .

• Biomarker Applications: pRPA2 T21 serves as a reliable biomarker of replication stress in formalin-fixed, paraffin-embedded (FFPE) tumor samples, with pRPA2 scores correlating with treatment response to platinum agents and PARP inhibitors .

How can researchers validate the specificity of Phospho-RPA2 (T21) antibody staining?

Ensuring antibody specificity is critical for reliable experimental outcomes. The following validation approaches are recommended:

• Peptide Competition Assays: Dot blot analysis comparing human RPA32/RPA2 (pT21) phospho peptide versus non-phospho peptide demonstrates high specificity of properly validated antibodies. In rigorous testing, antibodies should recognize the phosphorylated peptide but not the non-phosphorylated variant .

• RPA2 Knockdown Controls: siRNA-mediated knockdown of RPA2 results in significantly lower pRPA2 scores in cells, both with and without hydroxyurea treatment, confirming antibody specificity for the target protein .

• Phosphatase Treatment: Sample treatment with lambda phosphatase should eliminate signal from phospho-specific antibodies, providing evidence for phosphorylation-dependent recognition.

• Stress Induction Controls: Treatment of cells with increasing doses of hydroxyurea should result in a dose-dependent increase in pRPA2 signal, confirming the antibody's ability to detect stress-induced phosphorylation .

• Mutational Validation: Using cells expressing T21A mutant RPA2 as a negative control provides definitive evidence for antibody specificity, as these samples should show minimal signal .

What protocol optimizations are recommended for detecting Phospho-RPA2 (T21) in different sample types?

Protocol adjustments should be tailored to specific sample types:

For Cell Lysates (Western Blotting):

• Harvest cells in RIPA buffer containing phosphatase inhibitors

• Observe a specific band at approximately 32-40 kDa under reducing conditions

• Use dilutions between 1:1000 to 1:5000 depending on the specific antibody and treatment conditions

• Include positive controls (cells treated with 1 μM camptothecin for 1 hour)

For FFPE Tissue Sections (Immunofluorescence):

• Perform antigen retrieval (citrate buffer, pH 6.0) to expose epitopes

• Implement automated imaging analysis for consistent quantification

• Define pRPA2 score as the percentage of cells with ≥2 pRPA2 foci

• Classify samples as pRPA2-High if >16% of cells have ≥2 pRPA2 foci

For Dot Blot/ELISA:

• Use purified antibody at dilutions ranging from 1:1000 to 1:5000

• Block with 5% non-fat dry milk in TBST to minimize background

• Include both phosphorylated and non-phosphorylated peptide controls

How do different treatment conditions affect Phospho-RPA2 (T21) levels?

Various treatment conditions can modulate Phospho-RPA2 (T21) levels, which is important for experimental design:

Notably, cells expressing an S23A/S29A-RPA2 mutant show significant defects in T21 phosphorylation after DNA damage, indicating that prior phosphorylation at S23/S29 primes RPA2 for subsequent T21 phosphorylation .

How does Phospho-RPA2 (T21) status correlate with therapeutic responses in cancer?

Research has demonstrated significant correlations between RPA2 T21 phosphorylation and therapeutic responses in cancer, particularly ovarian cancer:

• Platinum Chemotherapy Response: HR-proficient, pRPA2-High tumors show significantly better responses to platinum chemotherapy than HR-proficient, pRPA2-Low tumors .

• Correlation with Drug Sensitivity: In patient-derived ovarian cancer cells, the number of pRPA2 foci significantly negatively correlates with carboplatin IC50 values (Pearson r= -0.90, P<0.001), indicating that higher phosphorylation levels predict better treatment sensitivity .

• PARP Inhibitor Efficacy: pRPA2-High tumors also demonstrate enhanced sensitivity to PARP inhibitors, potentially expanding the patient population that might benefit from these targeted therapies .

• Clinical Validation: These correlations have been validated across multiple patient cohorts, including both discovery (n=31) and validation cohorts (n=244) for platinum chemotherapy, and separate cohorts for PARP inhibitor treatment (n=87) .

These findings suggest that pRPA2 (T21) serves as a functional biomarker of replication stress that can predict therapeutic vulnerability independent of genetic markers.

What methodological approaches enable reliable assessment of Phospho-RPA2 (T21) in clinical samples?

Implementing pRPA2 (T21) as a clinical biomarker requires standardized methodological approaches:

• Standardized Scoring System:

  • Define pRPA2 score as the percentage of cells with ≥2 pRPA2 foci

  • Classify samples as pRPA2-High if >16% of cells have ≥2 pRPA2 foci

• Automated Quantification:

  • Employ automated imaging analysis to objectively measure pRPA2 foci

  • This approach reduces inter-observer variability and enables standardization across institutions

• Quality Control Measures:

  • Include positive controls (hydroxyurea-treated cell blocks)

  • Implement antibody validation steps for each batch of clinical testing

  • Ensure pathologist review for tumor content and sample quality

• Integration with Other Biomarkers:

  • Combine with RAD51 foci assessment for comprehensive HR proficiency evaluation

  • This integrated approach provides superior predictive value compared to either marker alone

How does RPA2 T21 phosphorylation influence mitotic progression after DNA damage?

RPA2 T21 phosphorylation plays a crucial role in regulating cellular responses to DNA damage during mitosis:

• Facilitation of Mitotic Exit:

  • RPA2 phosphorylation on T21 stimulates cells to exit from a damaged mitosis into a 2N G1 phase

  • Cells expressing T21A mutant RPA2 show significant defects in mitotic release after DNA damage (e.g., bleomycin treatment)

• Checkpoint Regulation:

  • T21A mutant cells exhibit extended spindle assembly checkpoint activation

  • These cells show higher percentage of cyclin B expression and BubR1 chromatin staining, indicating prolonged checkpoint signaling

• Cell Survival Impact:

  • Proper RPA2 phosphorylation increases cell viability following mitotic DNA damage

  • T21A mutant cells undergo apoptosis at higher rates (>27%) than wild-type cells (~6%) after DNA damage

• Pathway Dependencies:

  • ATM is required for mitotic RPA hyperphosphorylation after DNA damage

  • Chk1 activation and RPA hyperphosphorylation appear to be independent events, but Chk1 inhibition reduces mitotic exit

These findings highlight the importance of RPA2 T21 phosphorylation in coordinating cellular responses to DNA damage during mitotic progression.

What is the hierarchical relationship between different RPA2 phosphorylation sites?

RPA2 phosphorylation follows a well-defined hierarchical pattern with distinct functional implications:

• Initial Phosphorylation: CDK-mediated phosphorylation at S23 and S29 occurs during normal cell cycle progression, particularly during mitosis.

• Intermediate Phosphorylation: DNA damage triggers phosphorylation at S33 by ATR kinase as an early response.

• Late Phosphorylation: Severe replication stress or extensive DNA damage leads to phosphorylation at S4, S8, and lastly T21, primarily by DNA-PK or ATM.

The sequential nature of this phosphorylation cascade has been demonstrated experimentally:

• Expression of S23A/S29A-RPA2 mutant shows significant defects in hyperphosphorylation after DNA damage

• T21 is considered the last site to be phosphorylated and indicates impending replication catastrophe

• The T21A/S33A-RPA2 variant shows dramatically reduced cell viability under replication stress conditions

This hierarchical phosphorylation serves as a molecular "rheostat" that progressively shifts RPA2 function from DNA replication to DNA repair as damage severity increases.

How do different kinases contribute to RPA2 T21 phosphorylation?

Multiple kinases can phosphorylate RPA2 at T21 under different cellular conditions:

• DNA-PK: Primary kinase responsible for T21 phosphorylation following DNA double-strand breaks and severe replication stress.

• ATM: Critical for T21 phosphorylation during mitotic DNA damage, as demonstrated by the inability of ATM-deficient cells to support RPA hyperphosphorylation after mitotic DNA damage .

• ATR: Can phosphorylate T21 under conditions of prolonged replication stress, particularly when single-stranded DNA regions accumulate.

• Cyclin B-Cdc2 (CDK1): While primarily responsible for S23/S29 phosphorylation, it primes RPA2 for subsequent T21 phosphorylation by PIKK family kinases .

The involvement of multiple kinases creates redundancy in the system but also allows for context-specific phosphorylation patterns depending on the type and severity of DNA damage.

What contradictions exist in the current understanding of RPA2 T21 phosphorylation function?

Several unresolved questions and apparent contradictions exist in our current understanding:

• Repair vs. Catastrophe Signaling: T21 phosphorylation has been implicated both in facilitating DNA repair and in signaling impending replication catastrophe . This dual role suggests context-dependent functions that require further elucidation.

• Cell Survival Outcomes: While T21 phosphorylation facilitates mitotic exit after DNA damage and increases survival , it also correlates with enhanced sensitivity to DNA-damaging agents in cancer cells . This apparent contradiction may reflect differences between physiological DNA damage responses and therapeutic sensitization.

• Temporal Dynamics: The timing of T21 phosphorylation relative to other phosphorylation events may vary depending on the type of DNA damage and cellular context, complicating interpretations of its precise role.

• Interplay with Other Repair Pathways: The relationship between RPA2 T21 phosphorylation and homologous recombination repair shows complex dependencies that are not fully understood, particularly in HR-proficient versus HR-deficient contexts .

These contradictions highlight the complexity of RPA2 phosphorylation biology and underscore the need for further research to fully elucidate its context-dependent functions.

What are common technical challenges in detecting Phospho-RPA2 (T21) and their solutions?

Researchers frequently encounter several challenges when working with Phospho-RPA2 (T21) antibodies:

• Low Signal Intensity:

  • Challenge: Insufficient phosphorylation levels in unstressed cells

  • Solution: Pre-treat samples with DNA-damaging agents (1 μM camptothecin for 1 hour or 5 mM hydroxyurea for 24 hours )

• High Background in Immunofluorescence:

  • Challenge: Non-specific antibody binding

  • Solution: Optimize blocking with 5% non-fat dry milk in TBST; increase washing steps; test different antibody dilutions (typically 1:1000 to 1:5000)

• Cross-Reactivity Issues:

  • Challenge: Antibodies recognizing non-phosphorylated RPA2 or other phosphorylation sites

  • Solution: Validate specificity using dot blot analysis with phosphorylated and non-phosphorylated peptides ; include appropriate controls

• Inconsistent Results Between Experiments:

  • Challenge: Variability in phosphorylation induction

  • Solution: Standardize treatment conditions; include positive controls (hydroxyurea-treated samples) and negative controls (phosphatase-treated samples)

• Rapid Dephosphorylation During Sample Preparation:

  • Challenge: Loss of phosphorylation signal during processing

  • Solution: Include phosphatase inhibitors in all buffers; minimize processing time; consider immediate fixation

How can researchers differentiate specific from non-specific signals when analyzing Phospho-RPA2 (T21)?

Distinguishing specific from non-specific signals requires rigorous controls and validation approaches:

• Peptide Competition Assays:

  • Compare antibody recognition of the specific phospho-peptide versus non-phospho peptide

  • Dot blot analysis should show strong signal with the pT21 peptide but not with the non-phospho variant

• Mutant Controls:

  • Include samples from cells expressing T21A mutant RPA2, which should show minimal signal

  • The absence of signal in these samples confirms specificity for the phosphorylated residue

• Pattern Recognition:

  • Authentic pRPA2 (T21) signal typically appears as discrete nuclear foci in immunofluorescence

  • Diffuse nuclear staining or cytoplasmic signals often indicate non-specific binding

• Signal Induction:

  • Specific signal should increase in a dose-dependent manner with replication stress inducers

  • Treatment with hydroxyurea at increasing doses should result in corresponding increases in pRPA2 signal

• Molecular Weight Verification:

  • In Western blotting, authentic pRPA2 (T21) appears at approximately 32-40 kDa

  • Multiple bands at unexpected molecular weights suggest non-specific binding

What considerations are important when comparing Phospho-RPA2 (T21) levels across different experimental systems?

When comparing pRPA2 (T21) across different experimental systems, several factors must be considered:

• Baseline Phosphorylation Variations:

  • Different cell lines have varying baseline levels of replication stress and pRPA2 (T21)

  • Establish baseline levels for each cell line before making comparative analyses

• Cell Cycle Synchronization:

  • pRPA2 (T21) levels fluctuate throughout the cell cycle

  • Synchronize cells or incorporate cell cycle markers in analyses to ensure comparable populations

• Treatment Response Kinetics:

  • Different cell types may show varying temporal responses to DNA-damaging agents

  • Perform time-course experiments to identify optimal timepoints for each system

• Detection Method Standardization:

  • Use consistent antibody concentrations, incubation times, and detection methods

  • Include calibration standards when possible to enable quantitative comparisons

• Scoring System Adjustments:

  • The threshold for pRPA2-High classification (>16% of cells with ≥2 foci) was established in specific tumor types

  • This threshold may require adjustment for different experimental systems or cancer types

By addressing these considerations, researchers can make more reliable comparisons of pRPA2 (T21) levels across diverse experimental systems.

What potential exists for developing therapeutic strategies targeting RPA2 phosphorylation pathways?

Several promising therapeutic approaches targeting RPA2 phosphorylation pathways are being explored:

• Kinase Inhibitor Approaches:

  • Selective inhibition of kinases responsible for RPA2 T21 phosphorylation (DNA-PK, ATM, ATR)

  • Temporal modulation of these pathways might sensitize cells to DNA-damaging agents

• Synthetic Lethality Exploitation:

  • Identification of genetic contexts where RPA2 phosphorylation status creates specific vulnerabilities

  • Development of combination strategies targeting both replication stress and complementary pathways

• Biomarker-Guided Therapy:

  • Using pRPA2 (T21) levels to guide selection of platinum-based chemotherapy or PARP inhibitors

  • Development of adaptive treatment protocols based on dynamic changes in pRPA2 (T21) status

• Novel Combinations:

  • Exploration of synergistic combinations between agents that modulate RPA2 phosphorylation and established therapies

  • Investigation of sequencing strategies to maximize therapeutic efficacy

• Cell Cycle Checkpoint Modulator Combinations:

  • Given the role of pRPA2 (T21) in mitotic exit after DNA damage , combinations with checkpoint inhibitors (Chk1/2, ATR, WEE1) represent promising approaches

How might advanced imaging techniques enhance Phospho-RPA2 (T21) analysis in research and clinical settings?

Emerging imaging technologies offer significant potential for advancing pRPA2 (T21) analysis:

• Super-Resolution Microscopy:

  • Techniques like STORM or PALM could reveal the spatial organization of pRPA2 foci relative to other DNA repair factors

  • This could provide insights into the functional significance of various foci patterns

• Multiplexed Imaging:

  • Simultaneous detection of multiple DNA damage response markers alongside pRPA2 (T21)

  • This approach could elucidate the temporal relationship between different repair pathways

• Live-Cell Imaging:

  • Development of phospho-specific fluorescent reporters to track RPA2 phosphorylation dynamics in real-time

  • This would enable investigation of the temporal sequence of phosphorylation events

• Digital Pathology Integration:

  • Implementation of artificial intelligence algorithms for automated scoring of pRPA2 (T21) in clinical samples

  • This could improve standardization and efficiency in biomarker assessment

• Spatial Transcriptomics Correlation:

  • Integration of pRPA2 (T21) protein detection with spatial gene expression analysis

  • This could reveal transcriptional signatures associated with high pRPA2 regions

What unresolved questions remain regarding the fundamental biology of RPA2 T21 phosphorylation?

Despite significant advances, several fundamental questions about RPA2 T21 phosphorylation remain unresolved:

• Structural Consequences:

  • How does T21 phosphorylation alter the three-dimensional structure of RPA2?

  • What structural changes mediate the functional transition from replication to repair?

• Protein Interaction Landscape:

  • What is the comprehensive interactome of phosphorylated versus non-phosphorylated RPA2?

  • How do these interaction changes orchestrate the cellular response to DNA damage?

• Phosphorylation Dynamics:

  • What are the exact kinetics of T21 phosphorylation relative to other sites?

  • What phosphatases regulate T21 dephosphorylation and under what conditions?

• Tissue-Specific Functions:

  • Does the significance of T21 phosphorylation vary across different tissue types?

  • Are there tissue-specific factors that modulate RPA2 phosphorylation or its consequences?

• Evolutionary Conservation:

  • How conserved is the phosphorylation cascade across species?

  • Do orthologous phosphorylation sites in other organisms serve similar functions?

Addressing these questions will require interdisciplinary approaches combining structural biology, proteomics, advanced imaging, and systems biology to fully elucidate the complex biology of RPA2 T21 phosphorylation.