Phospho-RPA2 (Thr21) Antibody

What is RPA2 and why is its phosphorylation at Thr21 significant?

RPA2 (replication protein A 32 kDa subunit, also known as RFA2 or RPA p34) is a 32 kDa DNA-binding protein that forms part of the heterotrimeric RPA complex along with RPA1 (70 kDa) and RPA3 (14 kDa). This complex plays essential roles in DNA replication, recombination, and repair by binding to and stabilizing single-stranded DNA intermediates .

The phosphorylation of RPA2 at Thr21 occurs sequentially following phosphorylation at other sites and represents a critical event in cellular response to severe replication stress. Specifically, RPA2 becomes phosphorylated at multiple sites in response to DNA damage, with Thr21 being the last site to be phosphorylated, indicating impending replication catastrophe and irreparable damage . This phosphorylation event serves as a molecular switch that regulates RPA complex interactions with DNA repair and replication machinery.

How does the phosphorylation pattern of RPA2 change during DNA damage response?

During DNA damage response, RPA2 undergoes a complex pattern of phosphorylation at multiple sites:

RPA2 phosphorylation occurs through a complex priming and reciprocal priming mechanism involving cyclin-dependent kinase (CDK) and phosphatidylinositol kinase-related kinases (PIKKs), resulting in the mature hyperphosphorylated species of RPA32 . This sequential phosphorylation serves as a molecular timer, with Thr21 phosphorylation representing a critical threshold in the DNA damage response.

What cellular conditions induce phosphorylation of RPA2 at Thr21?

RPA2 Thr21 phosphorylation is specifically induced under the following conditions:

• Severe replication stress that leads to replication fork collapse

• Extended single-stranded DNA gaps formation

• Treatment with DNA damaging agents such as:

  • Camptothecin (CPT)

  • Hydroxyurea at high doses

  • Platinum-based chemotherapeutics

  • PARP inhibitors

• Irreparable DNA damage leading to replication catastrophe

Importantly, experimental evidence shows that the phosphorylation status of RPA2 at Thr21 correlates with the severity of replication stress, with increasing doses of hydroxyurea resulting in progressively higher pRPA2 scores in cancer cell lines .

What are the optimal techniques for detecting phospho-RPA2 (Thr21) in different sample types?

Several validated techniques can be employed to detect phospho-RPA2 (Thr21), each with specific applications and optimization requirements:

For detecting phospho-RPA2 (Thr21) foci in FFPE tumor samples, immunofluorescence has emerged as the preferred method as it enables quantitative assessment of replication stress through automated foci counting .

How should samples be prepared for optimal phospho-RPA2 (Thr21) detection?

Sample preparation is critical for reliable phospho-RPA2 (Thr21) detection:

For cell culture experiments:

• Induce DNA damage with appropriate agents (e.g., 1 µM Camptothecin for 1 hour or hydroxyurea at varying concentrations)

• For Western blot: Lyse cells in buffer containing phosphatase inhibitors

• For immunofluorescence on cultured cells: Fix with 4% paraformaldehyde and permeabilize with 0.2% Triton X-100

• For FFPE cell blocks: Treat cells with fixative, embed in paraffin, and section at 4-5 μm thickness

For tissue samples:

• Collect tissue samples with minimal ischemia time

• Fix immediately in 10% neutral buffered formalin for 24-48 hours

• Process, embed in paraffin, and section at 4-5 μm thickness

• For antigen retrieval: Use citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) under heat-induced conditions

The validation studies show that phospho-RPA2 (Thr21) antibody effectively detects RPA2 phosphorylation in both cultured cells and FFPE tumor samples with high specificity, as demonstrated by siRNA knockdown experiments .

What controls should be included when working with phospho-RPA2 (Thr21) antibodies?

Rigorous controls are essential for validating phospho-RPA2 (Thr21) antibody specificity:

Positive controls:

• HeLa or U2OS cells treated with 1 µM Camptothecin for 1 hour

• Cell lines treated with hydroxyurea at increasing concentrations (0.5-2 mM)

• γH2AX staining in parallel to confirm DNA damage induction

Negative controls:

• Untreated cell lines (should show minimal phospho-RPA2 (Thr21) staining)

• siRNA knockdown of RPA2 (should significantly reduce signal)

• Dephosphorylation of lysates with lambda phosphatase

• Blocking peptide competition assay

Internal controls for immunofluorescence:

• γH2AX staining to mark DNA damage sites

• DAPI staining for nuclear identification

• Cell cycle markers (e.g., PCNA) to identify S-phase cells

During analysis, samples should be assigned a pRPA2 score, defined as the percentage of cells with ≥2 or ≥5 pRPA2 foci, depending on the application context .

How can phospho-RPA2 (Thr21) serve as a biomarker for cancer treatment response?

Recent research has revealed that phospho-RPA2 (Thr21) has significant potential as a predictive biomarker for response to DNA-damaging therapies:

Key research findings:

• In ovarian cancer studies, pRPA2-High tumors showed significantly better responses to platinum chemotherapy than pRPA2-Low tumors

• The number of pRPA2 foci negatively correlated with carboplatin IC50 (Pearson r= -0.90, P<0.001) in patient-derived ovarian cancer cells

• HR-proficient tumors with high pRPA2 responded to platinum chemotherapy similarly to HR-deficient tumors

• pRPA2 foci can accurately identify which RAD51-High tumors will respond to PARP inhibitors

Clinical application methodology:

• Evaluate pRPA2-T21 foci via immunofluorescence in FFPE tumor samples

• Calculate pRPA2 scores (percentage of cells with ≥2 pRPA2 foci)

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

• Combine with RAD51 scoring for comprehensive HR proficiency assessment

• Use combined biomarker status to guide treatment decisions

This approach has been validated in multiple patient cohorts, including samples from patients treated with platinum chemotherapy (discovery cohort: n=31, validation cohort: n=244) and PARP inhibitors (n=87) .

What is the relationship between phospho-RPA2 (Thr21) and homologous recombination status?

The interplay between phospho-RPA2 (Thr21) and homologous recombination (HR) status has emerged as a critical determinant of treatment response:

Research findings:

Mechanistic explanation:
Replication stress, as indicated by pRPA2 foci, can sensitize HR-proficient tumors to DNA-damaging therapy through:

• Excessive ssDNA formation overwhelming HR repair capacity

• Fork collapse events leading to multiple double-strand breaks

• Impaired replication restart after treatment

This suggests that combining pRPA2 and RAD51 assessments provides a more comprehensive predictive biomarker than either alone .

How does cell cycle phase affect RPA2 phosphorylation patterns?

The phosphorylation of RPA2 at Thr21 and other sites is intimately linked to cell cycle progression:

Cell cycle-dependent phosphorylation patterns:

Experiments designed to study the phosphorylation status of RPA in S and G2 phase have demonstrated that:

• Cells reach mid-S phase about 3 hours after release from double thymidine block and G2 after about 7 hours

• RPA2 Ser4/Ser8 phosphorylation in G2 synchronized cells is necessary for increases in TopBP1 and Rad9 accumulation on chromatin

• RPA2 phosphorylation modulates ATM-dependent KAP-1 phosphorylation and Rad51 chromatin loading in G2 cells

Understanding these cell cycle-specific patterns is crucial for properly interpreting pRPA2 (Thr21) staining results in mixed cell populations.

What is the current understanding of kinase regulation of RPA2 Thr21 phosphorylation?

The regulation of RPA2 Thr21 phosphorylation involves multiple kinases in a complex signaling network:

Primary kinases targeting Thr21:

• ATR (ataxia telangiectasia and rad-3-related kinase) - primary kinase in replication stress

• ATM (ataxia telangiectasia mutated kinase) - contributes in response to double-strand breaks

• DNA-PKcs (DNA-dependent protein kinase catalytic subunit) - involved in non-homologous end joining contexts

Regulatory mechanisms:

• Sequential phosphorylation: Thr21 is typically phosphorylated after other sites, particularly after Ser33 phosphorylation by ATR

• Kinase priming: CDK-mediated phosphorylation at Ser23/Ser29 can prime RPA2 for subsequent phosphorylation at Thr21

• Feedback regulation: Hyperphosphorylated RPA2 can influence ATR activity, creating a potential feedback loop

Experimental approaches to study kinase specificity:

• Kinase inhibition studies using specific inhibitors for ATR (VE-821, AZD6738), ATM (KU-55933), and DNA-PKcs (NU7441)

• Phospho-site mutant analysis (e.g., T21A mutants)

• In vitro kinase assays with recombinant RPA2

• Chemical genetics approaches with analog-sensitive kinase mutants

Recent evidence suggests that the UV-induced, ATR-mediated inhibition of DNA replication specifically requires Thr21 and Ser33 phosphorylation, highlighting the crucial role of these sites among the numerous phosphorylation sites in the amino terminus of RPA2 .