Recombinant Chicken PCNA-interacting partner (PARPBP), partial

What is PARPBP and what is its fundamental role in cellular processes?

PARPBP (also known as PARI or C12orf48) is a protein containing a UvrD-like helicase domain that functions as a recombination inhibitor. Its primary role is to restrict unscheduled homologous recombination (HR) by interfering with the formation of RAD51-DNA HR structures. This protein is required for the preservation of genome stability in both human and chicken DT40 cells . PARPBP acts at replication forks, similar to the yeast Srs2 helicase, to prevent toxic recombination intermediates from forming during DNA replication. Unlike Srs2, which has both PCNA-dependent and independent functions, PARPBP appears to function exclusively through PCNA-dependent mechanisms .

How does the domain structure of PARPBP relate to its function?

PARPBP contains several key functional domains that dictate its activity:

• UvrD helicase-related domain: This is the core functional domain that shares similarity with yeast Srs2 and facilitates anti-recombinase activity

• PCNA-interacting protein box (PIP-box): Essential for binding to PCNA and localizing PARPBP to chromatin during S-phase

• SUMO-interacting motif (SIM): Enhances interaction with SUMO-modified PCNA

Research has demonstrated that both the PIP-box and SIM domains are critical for PARPBP function. Deletion studies show that PARPBP variants lacking either the PIP-box or the SIM fail to rescue the camptothecin sensitivity phenotype in PARPBP-deficient cells . The domain organization of PARPBP shows similarities with yeast Srs2, suggesting evolutionary conservation of anti-recombinase activity across species.

What methods are used to detect endogenous PARPBP in research settings?

Detection of endogenous PARPBP in cellular systems can be challenging due to its relatively low expression levels . Researchers typically employ several complementary approaches:

• Western blotting with specific anti-PARPBP antibodies

• Immunoprecipitation followed by mass spectrometry

• Chromatin fractionation assays to detect chromatin-bound PARPBP

• Immunofluorescence microscopy with optimized fixation protocols

When analyzing PARPBP localization during cell cycle progression, it's crucial to include appropriate controls for cell synchronization and to use specific markers for S-phase, as PARPBP is preferentially localized to chromatin during S-phase arrest but not during mitosis .

How does the PCNA interaction influence PARPBP activity and localization?

The interaction between PARPBP and PCNA is fundamental to PARPBP function. Experimental evidence indicates that PCNA serves as a critical cofactor that concentrates PARPBP at its sites of action, particularly at replication forks. This localization mechanism likely represents a regulatory control to prevent unconstrained PARPBP activity, which could be toxic to cells .

Key findings regarding this interaction include:

• PARPBP is preferentially localized to chromatin during S-phase

• The PIP-box is absolutely required for PCNA binding

• PCNA interaction-deficient PARPBP mutants show significantly impaired chromatin localization

• Wild-type human PARPBP can complement the loss of chicken PARPBP, but PIP-box mutants cannot

Biochemical studies using GST pulldowns and co-immunoprecipitation have demonstrated that full-length PARPBP directly interacts with PCNA, and this interaction is abolished when the C-terminal PIP-box is deleted . This PCNA-dependent recruitment mechanism ensures that PARPBP is specifically targeted to active replication sites where HR regulation is most critical.

What is the significance of SUMO modification in PARPBP-PCNA interactions?

SUMO modification plays an important regulatory role in the PARPBP-PCNA interaction. Research has shown that:

• PCNA is SUMOylated in human cells

• PARPBP interacts more strongly with SUMO1-modified PCNA than with unmodified PCNA

• The SUMO-interacting motif (SIM) in PARPBP is required for its function in vivo

This enhanced interaction with SUMOylated PCNA is reminiscent of yeast Srs2 recruitment to replication forks, where PCNA SUMOylation creates a binding platform for Srs2 . The preferential binding to SUMO-PCNA provides a mechanism for specific targeting of PARPBP activity to S-phase, when PCNA SUMOylation peaks. Mutations in the SIM domain of PARPBP result in functional defects similar to those observed with PIP-box mutations, highlighting the importance of both interaction interfaces.

How can researchers effectively study PARPBP-PCNA interactions in experimental systems?

To effectively study PARPBP-PCNA interactions, researchers should consider several methodological approaches:

• Protein interaction assays: GST pulldowns, co-immunoprecipitation, and yeast two-hybrid assays can be used to identify and characterize direct binding.

• PCNA SUMOylation analysis: To study the enhanced binding to SUMO-PCNA, researchers can use in vitro SUMOylation assays and create SUMO-fusion proteins.

• Live-cell imaging: Fluorescently-tagged PARPBP and PCNA can be used to track their co-localization during different cell cycle stages.

• Chromatin fractionation: This approach can determine if PARPBP mutants fail to localize to chromatin during S-phase, as demonstrated in previous studies where PCNA interaction-deficient PARPBP showed significantly reduced chromatin association .

• Domain-specific mutations: Creating specific mutations in the PIP-box and SIM domains allows for detailed analysis of their individual contributions to PARPBP function.

What is the molecular mechanism by which PARPBP inhibits homologous recombination?

PARPBP inhibits homologous recombination by interfering with the formation of RAD51-DNA structures, which are essential intermediates in the HR pathway . Unlike its yeast counterpart Srs2, which actively disassembles RAD51 nucleofilaments through ATP-driven helicase activity, PARPBP appears to function through a slightly different mechanism.

The molecular inhibition occurs through:

• Direct binding to RAD51, potentially preventing nucleofilament formation

• PCNA-dependent localization to replication forks, where it can intercept HR initiation

• Possible competition with HR factors for binding to DNA repair intermediates

This anti-recombinase activity is crucial for preventing inappropriate recombination events at replication forks, which could otherwise lead to genomic instability. Cell-based and biochemical assays have confirmed that PARPBP restricts unscheduled recombination by interfering with RAD51-DNA HR structure formation .

How does PARPBP deficiency affect HR-deficient cancer cells?

Interestingly, PARPBP deficiency has been shown to have protective effects in cells with HR deficiencies. Research has demonstrated that PARPBP knockdown suppresses the genomic instability of Fanconi Anemia/BRCA pathway-deficient cells . This suggests a synthetic rescue effect, where removal of an anti-recombinase partially compensates for defects in pro-recombination pathways.

This finding has important implications for cancer research, particularly for tumors with BRCA1/2 mutations. The mechanism likely involves:

• Removal of a restriction on the remaining HR capacity

• Allowing alternative HR sub-pathways to function more efficiently

• Potentially improving the limited recombination capacity of BRCA-deficient cells

These observations suggest that PARPBP could be a potential therapeutic target in cancers with specific DNA repair deficiencies, similar to how PARP inhibitors show synthetic lethality in BRCA-deficient cancers.

What are the differences between PARPBP function and Srs2 function in regulating HR?

While PARPBP and yeast Srs2 share functional similarities as anti-recombinases, several key differences exist:

A critical distinction is that Srs2 has detectable motor activity that actively dismantles RAD51 filaments, while PARPBP appears to work through a non-catalytic mechanism, likely requiring stoichiometric amounts at its sites of action . This fundamental difference suggests that PARPBP's regulation must be especially tight to prevent widespread HR inhibition.

What are effective methods for expressing and purifying recombinant chicken PARPBP?

Expressing and purifying recombinant chicken PARPBP presents several challenges due to its structural properties and relatively low natural expression levels. Based on published methodologies, researchers should consider:

• Expression systems:

  • Bacterial systems (E. coli BL21) with codon optimization for chicken sequences

  • Insect cell systems (Sf9, Hi5) for proteins requiring eukaryotic post-translational modifications

  • Mammalian expression systems for fully functional protein with native folding

• Purification tags and strategies:

  • N-terminal GST or His6 tags for affinity purification

  • Tandem affinity purification (TAP) for higher purity

  • Size exclusion chromatography as a final polishing step

• Solubility considerations:

  • Expression at lower temperatures (16-18°C) to improve folding

  • Addition of solubility enhancers (SUMO tag, MBP fusion)

  • Optimization of buffer conditions (salt concentration, pH, detergents)

When expressing partial PARPBP constructs, domain boundaries should be carefully designed based on sequence and structural analysis to ensure proper folding of individual domains.

What cell-based assays are most informative for studying PARPBP function?

Several complementary cell-based assays have proven valuable for elucidating PARPBP function:

• Homologous recombination reporter assays:

  • DR-GFP assay for measuring HR frequency

  • Sister chromatid exchange analysis

  • RAD51 foci formation assays by immunofluorescence

• DNA damage sensitivity assays:

  • Clonogenic survival assays following camptothecin treatment

  • Sensitivity to other DNA-damaging agents (hydroxyurea, mitomycin C)

  • PARPBP-deficient DT40 cells show hypersensitivity to camptothecin, which can be rescued by wild-type human PARPBP but not by PIP-box or SIM mutants

• Protein localization studies:

  • Chromatin fractionation to assess chromatin association

  • Immunofluorescence microscopy throughout cell cycle

  • Live-cell imaging with fluorescently tagged proteins

• Genetic complementation:

  • PARPBP knockout chicken DT40 cell complementation with wild-type or mutant constructs

  • CRISPR/Cas9-mediated gene editing for creating specific mutations

These assays, particularly when used in combination, can provide comprehensive insights into PARPBP function in regulating homologous recombination and maintaining genomic stability.

How can researchers effectively analyze PARPBP interactions with binding partners?

To thoroughly characterize PARPBP interactions with its binding partners (PCNA, RAD51, PARP-1), researchers should employ a multi-faceted approach:

• In vitro binding assays:

  • GST pulldown assays with purified components

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Cellular interaction studies:

  • Co-immunoprecipitation from cell extracts

  • Proximity ligation assay (PLA) for detecting protein interactions in situ

  • FRET/BRET approaches for real-time interaction analysis

• Domain mapping:

  • Truncation and point mutation analysis to identify critical residues

  • Peptide competition assays to validate binding interfaces

  • Crosslinking mass spectrometry to identify interaction sites

• Functional validation:

  • Complementation assays with interaction-deficient mutants

  • Analysis of the effect of mutations on chromatin localization

  • Assessment of the impact on DNA damage sensitivity

Previous research has successfully used GST pulldowns and co-immunoprecipitation to demonstrate that full-length PARPBP interacts with PCNA from extracts of HeLa cells, and that this interaction requires the PIP-box domain .

How is PARPBP expression correlated with cancer progression and prognosis?

Research has identified significant associations between PARPBP expression and cancer progression, particularly in breast cancer. Analysis of clinical data has revealed:

• High PARPBP expression is significantly associated with:

  • Advanced tumor status (T2-T4)

  • Lymph node metastasis (N1-N3)

  • Advanced TNM stage (III-IV)

The following table summarizes the correlation between PARPBP expression and clinical parameters in breast cancer patients:

These correlations suggest that PARPBP may serve as a prognostic marker in breast cancer, with high expression associated with more advanced disease and potentially poorer outcomes .

What is the relationship between PARPBP and chemotherapy resistance?

PARPBP has been implicated in chemotherapy resistance, particularly to anthracycline-based treatments in breast cancer. Research findings indicate:

• PARPBP confers anthracycline resistance in breast cancer cells

• This resistance mechanism likely involves enhanced DNA repair capabilities

• Anthracycline antibiotics cause DNA damage by embedding between DNA double-stranded bases, but tumor cells with elevated PARPBP may more effectively counter this damage

The underlying mechanisms may involve:

• PARPBP's function in regulating homologous recombination

• Interaction with other DNA repair proteins, including PARP-1, PCNA, and RAD51

• Possible activation of alternative DNA repair pathways, such as translesion synthesis (TLS) and non-homologous end joining (NHEJ)

Understanding PARPBP's role in chemoresistance could potentially lead to the development of new chemosensitizers or biomarkers for predicting treatment response in breast cancer patients.

How might targeting PARPBP improve cancer therapy outcomes?

Targeting PARPBP represents a promising therapeutic strategy, particularly for specific cancer subtypes:

• Potential synergy with existing therapies:

  • PARP inhibitors: Research suggests potential synthetic lethal interactions between PARPBP and PARP inhibition pathways

  • DNA-damaging chemotherapies: PARPBP downregulation might sensitize cancer cells to these agents

  • Radiation therapy: Given PARPBP's role in DNA repair, targeting it might enhance radiosensitivity

• Specific cancer contexts:

  • HR-deficient cancers: PARPBP downregulation has been shown to suppress genomic instability in Fanconi Anemia/BRCA pathway-deficient cells

  • Anthracycline-resistant tumors: Inhibiting PARPBP might restore sensitivity

  • Breast cancers with high PARPBP expression: These might be particularly susceptible to PARPBP-targeting approaches

• Potential targeting approaches:

  • Small molecule inhibitors targeting the UvrD-like domain

  • Peptide inhibitors disrupting PCNA-PARPBP interaction

  • RNA interference or antisense oligonucleotides to reduce expression

A particularly interesting finding is that PARPBP overexpressing ER+ breast cancers might be sensitive to platinum-based compounds and/or radiotherapy , suggesting potential for treatment stratification based on PARPBP status.

How does PARPBP coordinate with other DNA repair mechanisms?

PARPBP functions within a complex network of DNA repair mechanisms, and its interplay with other pathways represents an important area for advanced research:

• Interactions with translesion synthesis (TLS):

  • PARPBP has been shown to activate error-prone DNA repair pathways such as TLS when HR is inhibited

  • This could result in mutagenesis and genomic instability, potentially contributing to cancer progression or treatment resistance

• Coordination with non-homologous end joining (NHEJ):

  • PARPBP may influence NHEJ activity, another error-prone pathway

  • The balance between HR and NHEJ regulation by PARPBP warrants further investigation

• PARP-1 interaction implications:

  • Direct interaction between PARPBP and PARP-1 has been reported

  • This suggests potential crosstalk between PARP-mediated and PARPBP-mediated repair regulation

  • Combined targeting of both pathways might enhance therapeutic efficacy

• Cell cycle-specific regulation:

  • PARPBP's S-phase-specific chromatin localization suggests coordination with cell cycle checkpoints

  • Research into how PARPBP activity is regulated throughout the cell cycle could reveal additional therapeutic vulnerabilities

Understanding these complex interactions will require sophisticated experimental approaches and may reveal novel therapeutic strategies for cancers with specific DNA repair defects.

What are the evolutionary implications of PARPBP conservation across species?

The evolutionary conservation of PARPBP across species raises important questions about its fundamental role in genome maintenance:

• Functional conservation vs. divergence:

  • While PARPBP shares functional similarities with yeast Srs2, there are notable differences in mechanism

  • Comparative studies across species could reveal how anti-recombinase functions have evolved

• Species-specific adaptations:

  • Chicken PARPBP may have unique properties compared to human PARPBP

  • Understanding these differences could provide insights into species-specific DNA repair regulation

• Structural conservation:

  • The UvrD-like domain, PIP-box, and SIM are conserved features

  • Phylogenetic analyses show that PARPBP closely clusters with family members from multiple species

  • Structural comparison across species could identify critical functional elements

• Evolutionary pressure:

  • The conservation of anti-recombinase function suggests strong evolutionary pressure to maintain this activity

  • This highlights the critical importance of properly regulating HR during DNA replication

These evolutionary perspectives can inform both basic understanding of PARPBP function and potential therapeutic applications targeting this protein.

What methodological advances are needed to better study PARPBP function in vitro and in vivo?

Advancing PARPBP research will require several methodological innovations:

• Improved protein expression and purification:

  • Development of expression systems that yield higher amounts of functional protein

  • Structural biology approaches (cryo-EM, X-ray crystallography) to elucidate PARPBP structure

• Real-time single-molecule studies:

  • Direct visualization of PARPBP action on DNA substrates

  • Analysis of how PARPBP affects RAD51 nucleofilament formation in real-time

• In vivo models:

  • Development of conditional knockout mouse models for tissue-specific PARPBP deletion

  • CRISPR-engineered cellular models with specific PARPBP mutations or domain deletions

  • Patient-derived xenografts to study PARPBP function in human tumors

• High-throughput screening approaches:

  • Screens for small molecule inhibitors of PARPBP

  • Synthetic lethality screens to identify genetic interactions

• Integration of multi-omics data:

  • Combining genomics, transcriptomics, and proteomics to understand PARPBP regulation

  • Systems biology approaches to model PARPBP in the context of DNA repair networks

These methodological advances would significantly enhance our understanding of PARPBP function and potentially accelerate the development of therapeutic strategies targeting this protein.