Phospho-PRKDC (S2056) Recombinant Monoclonal Antibody

What is the biological function of PRKDC and significance of S2056 phosphorylation?

PRKDC functions as a molecular sensor for DNA damage and plays a crucial role in DNA non-homologous end joining (NHEJ) required for double-strand break (DSB) repair and V(D)J recombination. The phosphorylation at S2056 occurs specifically in response to double-stranded DNA breaks and ATM activation. This post-translational modification is part of the S2056 (PQR) autophosphorylation cluster, which has significant implications for PRKDC function in DNA repair pathways .

Autophosphorylation at S2056 has been demonstrated to affect DNA-PK kinase activity and NHEJ ability, with evidence suggesting it contributes to the regulation of DNA repair machinery assembly and disassembly. The physiological significance of S2056 cluster phosphorylation includes ensuring proper chromosomal NHEJ processes and suppressing excessive end-resection during DNA repair .

What applications are Phospho-PRKDC (S2056) antibodies validated for?

Phospho-PRKDC (S2056) antibodies have been rigorously validated for multiple experimental applications:

These applications allow researchers to accurately assess PRKDC phosphorylation levels across various experimental conditions and cell types .

What are suitable positive controls for validating Phospho-PRKDC (S2056) antibody performance?

HeLa cells have been identified as reliable positive controls for Phospho-PRKDC (S2056) antibody validation. For induction of phosphorylation, cells should ideally be treated with DNA-damaging agents such as ionizing radiation, etoposide, or bleomycin, which activate the DNA damage response pathway and induce PRKDC phosphorylation at S2056. The baseline phosphorylation can be compared to treated conditions to verify antibody specificity and sensitivity .

How can researchers distinguish between ATM-dependent and DNA damage-dependent phosphorylation of PRKDC at S2056?

Distinguishing between ATM-dependent and direct DNA damage-dependent phosphorylation requires careful experimental design:

• ATM inhibition approach: Pretreat cells with specific ATM inhibitors (e.g., KU-55933) before inducing DNA damage. Compare phosphorylation levels at S2056 between ATM-inhibited and uninhibited samples using the Phospho-PRKDC (S2056) antibody.

• Genetic approach: Use ATM-knockout or ATM-depleted cell lines alongside wild-type controls to assess S2056 phosphorylation levels after DNA damage induction.

• Time-course analysis: ATM-dependent phosphorylation typically follows different kinetics than direct DNA damage-induced phosphorylation. Perform time-course experiments after DNA damage to capture these differences.

• Phosphorylation site mutants: Generate S2056A mutant constructs to confirm specificity of signals detected by the antibody.

Research has established that phosphorylation at S2056 occurs specifically in response to double-stranded DNA breaks and requires ATM activation, providing a framework for distinguishing these pathways .

What is the relationship between PRKDC S2056 phosphorylation and NHEJ efficiency?

The S2056 (PQR) cluster phosphorylation plays a crucial role in the efficiency of NHEJ repair:

• End-ligation facilitation: The PQR cluster phosphorylation contributes to the end-ligation step of chromosomal NHEJ, particularly in contexts where XLF (XRCC4-like factor) is deficient. Loss of DNA-PKcs S2056 cluster phosphorylation in XLF-deficient models severely compromises both B and T lymphocyte development at the V(D)J recombination stage .

• Prevention of excessive resection: Studies of PQR/PQR DNA-PKcs mutants in XLF-deficient cells reveal large deletions in coding joints (CJs) and signal joints (SJs), indicating that S2056 cluster phosphorylation suppresses excessive end-resection during NHEJ .

• Functional redundancy: The PQR cluster appears to have partially overlapping functions with XLF in promoting NHEJ, suggesting evolutionary redundancy in the DNA repair machinery .

• Temporal regulation: DNA-PK preferentially phosphorylates substrates before it autophosphorylates at sites including S2056, suggesting that this autophosphorylation may regulate the disassembly of the DNA repair complex after repair completion .

Researchers studying this relationship should consider implementing assays that measure both phosphorylation status and repair efficiency simultaneously, such as combined immunofluorescence and neutral comet assays.

How does PRKDC activation relate to PKMYT1 inhibition strategies in cancer research?

Recent genome-wide CRISPR screens have identified an important relationship between PRKDC activation and sensitivity to PKMYT1 inhibition in pancreatic ductal adenocarcinoma (PDAC):

• Synthetic lethality: PRKDC activation promotes PKMYT1 inhibition-induced γH2AX accumulation and cytotoxicity in PDAC cells, suggesting a potential synthetic lethal relationship that could be therapeutically exploited .

• PRKDC dependence: PRKDC knockdown using shRNAs conferred resistance to the PKMYT1 inhibitor RP-6306 in PDAC cells, indicating that PRKDC activity is essential for the efficacy of PKMYT1 inhibition in these cancer cells .

• Methodological considerations: When designing experiments to investigate this relationship, researchers should:

  • Use both genetic (shRNA) and pharmacological approaches to modulate PRKDC activity

  • Measure phosphorylation at S2056 as a marker of PRKDC activation

  • Assess downstream markers including γH2AX accumulation

  • Evaluate cytotoxicity through multiple assays (e.g., cell viability, apoptosis markers)

• Clinical relevance: The activation status of PRKDC (potentially measured via S2056 phosphorylation) could serve as a biomarker for patient stratification in clinical applications of PKMYT1 inhibitors .

What optimization strategies can improve detection specificity with Phospho-PRKDC (S2056) antibodies?

To optimize experimental protocols for maximum specificity and sensitivity:

• Phosphatase inhibitors: Always include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers to preserve phosphorylation status.

• Blocking optimization: For Western blot applications, compare BSA vs. non-fat dry milk as blocking agents, as phospho-epitopes can sometimes be masked by milk proteins.

• Signal amplification: For detecting low levels of phosphorylated PRKDC, consider using HRP-conjugated secondary antibodies with enhanced chemiluminescence substrates or tyramide signal amplification for immunostaining.

• Antibody validation controls:

  • Phosphatase treatment control: Treat one sample with lambda phosphatase to remove phosphorylation and confirm antibody specificity

  • Phospho-null mutant (S2056A) as negative control

  • DNA damage-induced samples as positive controls

• Dilution optimization: Perform careful titration experiments using the manufacturer's recommended ranges:

  • WB: 1:500 - 1:2000

  • IHC-P: 1:100 - 1:500

  • IF/ICC: 1:50 - 1:200

What are the advantages of recombinant monoclonal antibodies for phospho-PRKDC detection?

Recombinant monoclonal antibodies offer several advantages over traditional polyclonal antibodies for phospho-PRKDC detection:

• Superior lot-to-lot consistency: The recombinant production method ensures consistent antibody performance across different lots, which is critical for longitudinal studies of PRKDC phosphorylation .

• Continuous supply: The recombinant production process guarantees uninterrupted availability of antibodies with identical binding properties, ensuring experimental reproducibility .

• Animal-free manufacturing: Recombinant antibodies reduce ethical concerns associated with animal immunization and provide a more sustainable source of research reagents .

• Enhanced specificity: Monoclonal antibodies target a single epitope, reducing cross-reactivity with other phosphorylation sites on PRKDC or related proteins.

• Reduced background: The high specificity of recombinant monoclonal antibodies typically results in cleaner signals in applications such as immunohistochemistry and immunofluorescence.

How can Phospho-PRKDC (S2056) antibodies be used to study DNA damage response pathways?

Phospho-PRKDC (S2056) antibodies serve as valuable tools for investigating various aspects of DNA damage response:

• Spatiotemporal dynamics: Use time-course experiments with immunofluorescence to track the recruitment and phosphorylation of PRKDC at DNA damage sites. Phosphorylated DNA-PK co-localizes with γH2A.X and 53BP1 at sites of DNA damage, allowing for colocalization studies of repair complex assembly .

• Pathway crosstalk: Analyze the relationship between PRKDC phosphorylation and other DNA damage response proteins such as ATM, ATR, and BRCA1/2 by combinatorial immunostaining or Western blotting.

• Therapeutic response monitoring: Evaluate changes in S2056 phosphorylation in response to DNA-damaging cancer therapies (radiation, chemotherapy) to predict treatment efficacy.

• Genetic screening validation: In CRISPR screens or genetic studies, use phospho-specific antibodies to confirm the impact of identified genes on PRKDC activation.

• Quantitative phosphoproteomics: Combine immunoprecipitation using Phospho-PRKDC (S2056) antibodies with mass spectrometry to identify interacting partners specific to the phosphorylated form.

What is the significance of PRKDC S2056 phosphorylation in cancer research and therapy development?

PRKDC S2056 phosphorylation has important implications for cancer research and therapeutic strategies:

• Biomarker potential: S2056 phosphorylation status can serve as a biomarker for:

  • DNA damage repair capacity in tumors

  • Resistance to radiotherapy and certain chemotherapeutics

  • Efficacy of PRKDC inhibitors and other DNA repair-targeting drugs

• Therapeutic targeting: The relationship between PRKDC activation and sensitivity to PKMYT1 inhibitors in PDAC highlights the importance of PRKDC phosphorylation status in predicting therapeutic responses. This suggests that measuring S2056 phosphorylation could help stratify patients for PKMYT1 inhibitor treatment .

• Synthetic lethality approaches: Understanding the contexts where PRKDC activation (measured via S2056 phosphorylation) creates vulnerabilities can inform the development of synthetic lethal therapeutic strategies, particularly in cancers with specific genetic backgrounds such as TP53 loss .

• Resistance mechanisms: Changes in S2056 phosphorylation patterns may indicate adaptive responses to therapies, providing insights into resistance mechanisms.

What are the latest research findings regarding the functional significance of S2056 cluster phosphorylation?

Recent research has revealed important insights about the S2056 phosphorylation cluster:

• End-ligation role: Despite previous studies showing limited phenotypes in S2056 cluster alanine substitution models, recent work using XLF-deficient backgrounds has revealed that S2056 cluster phosphorylation plays a critical role in the end-ligation step of chromosomal NHEJ .

• Suppression of end-resection: Analysis of coding joints and signal joints from cells with mutations in the S2056 cluster (PQR/PQR DNA-PKcs) reveals that this phosphorylation is essential for suppressing excessive end-resection during DNA repair .

• Functional redundancy: The S2056 cluster appears to have partially overlapping functions with XLF in promoting NHEJ, explaining why single mutations often show limited phenotypes. This functional redundancy suggests evolutionary mechanisms to ensure robust DNA repair .

• Connection to PKMYT1 inhibition pathways: Recent studies have connected PRKDC activation (including S2056 phosphorylation) to the efficacy of PKMYT1 inhibitors in cancer treatment, expanding the therapeutic relevance of this phosphorylation site .