UVSSA Antibody, Biotin conjugated

What is UVSSA and why is it important in DNA repair research?

UVSSA is a critical component of the transcription-coupled nucleotide excision repair (TC-NER) pathway. It serves two essential functions: stabilizing Cockayne syndrome group B (CSB) protein by recruiting the deubiquitinating enzyme USP7, and promoting TFIIH recruitment through direct interactions . UVSSA's importance extends beyond UV damage repair to include transcription-coupled repair of interstrand crosslinks (ICLs) . Recent research demonstrates that UVSSA expression positively correlates with ICL chemotherapy resistance in human cancer cell lines, highlighting its potential significance in cancer research and therapeutic development .

What cellular localization patterns should be expected when using UVSSA antibodies in immunofluorescence studies?

When using biotin-conjugated UVSSA antibodies for immunofluorescence, researchers should expect primarily nuclear localization with increased chromatin association following DNA damage. After UV irradiation or ICL-inducing treatment, UVSSA localizes to chromatin through its interactions with the transcription machinery, specifically with RNA polymerase II, CSA, CSB, and TFIIH . Different patterns may emerge depending on damage type - with UV damage causing more diffuse nuclear staining and ICL damage potentially showing more focal accumulation . Importantly, UVSSA's localization is dependent on CSB, as studies have shown that the UV-induced interaction between UVSSA and other TC-NER factors is lost in CSB-deficient cells .

How can researchers verify the specificity of UVSSA antibodies in experimental systems?

Verification of UVSSA antibody specificity requires multiple complementary approaches:

• Genetic controls: Compare antibody signals between wild-type cells and UVSSA knockout/knockdown cells generated through CRISPR-Cas9 or RNAi techniques. Single cell cloning methodologies using dilutions of approximately 4.8 cells per ml distributed across 96-well plates can be employed to isolate pure UVSSA-deficient cell populations .

• Western blot validation: Identify the expected band pattern, including both unmodified UVSSA (approximately 81 kDa) and its modified forms, particularly the mono-ubiquitinated form (UVSSA-2) which appears as a distinct higher molecular weight band .

• Immunoprecipitation controls: Use cells expressing tagged versions of UVSSA, such as FLAG-UVSSA or mScarletI-HA-tagged UVSSA from its endogenous locus, to confirm antibody specificity through parallel detection methods .

• Cross-reactivity assessment: Test the antibody against recombinant UVSSA and related proteins to ensure minimal cross-reactivity with other TC-NER components.

What is the significance of UVSSA's ubiquitination status in experimental design?

UVSSA exists in multiple ubiquitination states, which significantly impacts experimental design considerations. Research has identified distinct forms including native UVSSA (UVSSA-1), mono-ubiquitinated UVSSA (UVSSA-2), and multiple poly-ubiquitinated forms (UVSSA-3 through UVSSA-6) . Mono-ubiquitination occurs primarily at lysine 414, which has been identified as the sole target of ubiquitination .

When designing experiments, researchers should consider:

• Extraction conditions may affect detection of different UVSSA forms, as ubiquitinated UVSSA species are largely associated with insoluble chromatin fractions .

• Chromatin fractionation approaches using specialized buffers (such as Lysis Buffer NPI-10-Ig containing Benzonase and 300 mM NaCl) are recommended for stripping tightly chromatin-associated modified UVSSA forms .

• UV irradiation changes the distribution of different UVSSA forms, with evidence of mild reduction in UVSSA-1 and UVSSA-2 intensities following UV treatment .

How can biotin-conjugated UVSSA antibodies be optimized for chromatin immunoprecipitation (ChIP) assays?

Optimizing biotin-conjugated UVSSA antibodies for ChIP requires addressing several technical considerations:

• Chromatin preparation: UVSSA exists in different sub-nuclear fractions, with ubiquitinated forms predominantly in insoluble chromatin . Effective chromatin preparation requires protocols that can release UVSSA from these tightly bound fractions.

• Crosslinking optimization: Standard formaldehyde crosslinking (1%) for 10 minutes at room temperature may be insufficient for capturing transient UVSSA interactions. Consider dual crosslinking approaches using protein-protein crosslinkers (DSP or EGS) followed by formaldehyde.

• Enrichment strategy: Utilize the biotin conjugation for streptavidin-based pull-down, which offers higher affinity and specificity than traditional antibody-based methods. For complex containing both UVSSA and CSB, sequential ChIP (re-ChIP) may be necessary due to their dynamic interaction patterns following DNA damage .

• Validation controls: Include controls that account for the biphasic response of UVSSA levels after UV irradiation, as research has demonstrated that both CSB and UVSSA exhibit a biphasic decrease and recovery upon UV irradiation .

• Sonication conditions: Modified UVSSA species may require adapted sonication protocols to ensure proper chromatin fragmentation while preserving protein-DNA interactions.

What methodological approaches can resolve contradictory data when studying UVSSA-USP7 interactions?

Resolving contradictory data regarding UVSSA-USP7 interactions requires systematic methodological approaches:

• Delineate direct versus indirect interactions: While UVSSA interacts with USP7, evidence suggests that USP7 specifically deubiquitinates CSB but not UVSSA . To clarify conflicting results:

  • Perform in vitro deubiquitination assays using purified components

  • Compare native versus recombinant protein interactions

  • Use domain-specific mutations to map interaction regions

• Address cellular context variations: The research demonstrates that USP7 physically interacts with both CSB and VCP/p97, with UV radiation enhancing the USP7-VCP/p97 association . These context-dependent interactions may explain contradictory findings.

• Evaluate temporal dynamics: The biphasic response of UVSSA after DNA damage suggests time-dependent interactions that may yield contradictory results if sampled at different timepoints .

• Compare subcellular fractions: Different results may emerge depending on whether analyses are performed on soluble chromatin fractions versus insoluble chromatin fractions, where the distribution of UVSSA forms differs significantly .

• Control for protein overexpression artifacts: Studies have shown that CSB overexpression stabilizes UVSSA but decreases UVSSA's presence in nuclease-releasable/soluble chromatin while increasing ubiquitinated UVSSA in insoluble chromatin .

How can researchers differentiate between UVSSA functions in TC-NER versus transcription-coupled interstrand crosslink repair (TC-ICR)?

Differentiating UVSSA's roles in TC-NER versus TC-ICR requires specialized experimental designs:

• Damage-specific induction: Use targeted approaches to induce specific types of DNA damage:

  • UV irradiation predominantly causes pyrimidine dimers and 6-4 photoproducts

  • ICL-inducing agents (cisplatin, MMC, SJG-136) create interstrand crosslinks

• Reporter system selection: Employ specialized reporter systems that distinguish between repair pathways:

  • For TC-ICR specifically, use fluorescence-based reporters lacking an origin of replication to prevent replication-coupled repair mechanisms

  • Control for replication status in dividing cells, as replication-coupled ICL repair can confound results

• Temporal resolution experiments: The repair kinetics differ between TC-NER and TC-ICR, with TC-ICR generally requiring longer timeframes for resolution .

• Genetic dissection approaches: Systematically inactivate components specific to each pathway:

  • FANCM/FAAP24 are more specific to replication-coupled ICL repair

  • CSB is required for both TC-NER and TC-ICR

• Interaction partner analysis: Identify differential interaction partners through techniques like BioID or proximity ligation assays to distinguish pathway-specific protein complexes.

What are the critical technical considerations when analyzing UVSSA-protein interactions using biotin-conjugated antibodies?

When analyzing UVSSA-protein interactions with biotin-conjugated antibodies, researchers should address these critical technical considerations:

• Biotin interference assessment: The biotin conjugation may sterically hinder interaction sites, particularly at the VHS domain of UVSSA which creates a binding interface of 392.3 Ų with partner proteins like STK19 . Alternative labeling approaches should be considered for comparison.

• Complex stability preservation: The UVSSA interactions with TC-NER factors are highly dependent on the presence of a complete TC-NER complex. Research demonstrates that in the absence of CSB, CSA, or other components, these interactions can be completely lost . Extraction conditions must preserve these delicate protein complexes.

• Domain-specific interaction mapping: Consider that different domains of UVSSA mediate specific interactions:

  • The VHS domain creates specific hydrophobic interactions with partners

  • Different domains may be responsible for interactions with USP7 versus TFIIH

• Chromatin context: Many UVSSA interactions occur specifically in the chromatin context. Studies have shown that ubiquitinated UVSSA forms are largely associated with insoluble chromatin in human cells, requiring specialized extraction protocols .

• Detection sensitivity optimization: The biphasic response of UVSSA levels following DNA damage necessitates highly sensitive detection methods that can track changes in both protein levels and modification states over time .

What extraction protocols best preserve UVSSA ubiquitination states for subsequent analysis?

Preserving UVSSA ubiquitination states requires specialized extraction protocols:

Research has demonstrated that insoluble chromatin fractions contain the highest abundance of ubiquitinated UVSSA forms, with multiple bands of UVSSA larger than its calculated molecular weight appearing at much higher intensity in these fractions . For optimal preservation, samples should be processed rapidly with deubiquitinase inhibitors (e.g., N-ethylmaleimide) and proteasome inhibitors (e.g., MG132) included in all buffers .

How can researchers quantitatively assess UVSSA involvement in transcription recovery after DNA damage?

Quantitative assessment of UVSSA's role in transcription recovery requires multifaceted approaches:

What controls are essential when evaluating UVSSA-dependent deubiquitination in experimental systems?

When evaluating UVSSA-dependent deubiquitination, these controls are essential:

• USP7 catalytic activity controls: Include both wild-type USP7 and catalytically dead (CD) USP7 containing a cysteine to serine substitution at amino acid 223, which specifically inactivates the deubiquitinating activity . Research has shown that both WT and CD USP7 associate with CSB, but only WT USP7 deubiquitinates CSB .

• Substrate specificity controls: Comparative analysis of CSB versus UVSSA as deubiquitination substrates, as research has demonstrated that USP7 specifically deubiquitinates CSB but not UVSSA in vitro .

• Chromatin fraction controls: Analyze both soluble and insoluble chromatin fractions separately, as ubiquitinated forms show differential distribution. Western blotting analysis has revealed that <250 kDa CSB-Ub conjugates exist in both chromatin fractions, while >250 kDa CSB-Ub conjugates exist primarily in insoluble chromatin fractions .

• Time-course controls: Account for the biphasic response patterns observed after DNA damage, with both CSB and UVSSA exhibiting a decrease followed by recovery upon UV irradiation .

• Genetic background validation: Include UVSSA-deficient and USP7-deficient cells to establish dependency relationships. Single cell cloning methodologies can be employed to generate these controls through isolation of pure deficient cell populations .

How can researchers accurately determine the stoichiometry of UVSSA interactions in multi-protein complexes?

Determining accurate stoichiometry of UVSSA in multi-protein complexes requires integrated approaches:

• Quantitative mass spectrometry: Apply techniques like SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling combined with absolute quantification using synthetic peptide standards.

• Single-molecule imaging: Utilize techniques like photobleaching step analysis with fluorescently tagged proteins to count discrete subunits within complexes.

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS): Determine absolute molecular masses of intact complexes to infer component stoichiometry.

• Native mass spectrometry: Analyze intact protein complexes to determine precise molecular composition and stoichiometry without disrupting native interactions.

• Cross-linking mass spectrometry (XL-MS): Identify specific interaction interfaces and validate structural models. Research has identified specific interaction domains, such as the β-turn from residue 202-205 on the WH-C domain of STK19 that interacts with the VHS domain of UVSSA, with Tyr204 inserting into a hydrophobic cavity created by helices α1 and α3 .

What emerging technologies might enhance detection sensitivity and specificity for UVSSA studies?

Several emerging technologies hold promise for advancing UVSSA research:

• Proximity-dependent biotinylation: BioID or TurboID approaches can identify transient UVSSA interaction partners that may be missed by traditional co-immunoprecipitation techniques.

• Super-resolution microscopy: Techniques like STORM, PALM, or expansion microscopy can resolve UVSSA localization with nanometer precision, potentially revealing previously undetectable spatial patterns following DNA damage.

• CRISPR-based genomic tagging: Endogenous tagging strategies eliminate overexpression artifacts that have been shown to alter UVSSA distribution and interaction patterns .

• Single-cell protein analysis: Mass cytometry or microfluidic antibody capture techniques can reveal cell-to-cell variability in UVSSA levels and modifications that may be masked in population averages.

• Cryo-electron microscopy: Structural determination of UVSSA-containing complexes can provide mechanistic insights into its precise role in coordinating repair factors, building on recent findings about specific interaction interfaces .