Recombinant Mouse DNA excision repair protein ERCC-6-like (Ercc6l), partial

What is Mouse DNA Excision Repair Protein ERCC-6-Like (Ercc6l)?

Mouse DNA excision repair protein ERCC-6-like (Ercc6l) is a specialized protein involved in DNA damage repair mechanisms, similar to its human ortholog ERCC6. It plays a crucial role in maintaining genomic integrity by participating in repair processes when genes undergoing transcription become damaged or inoperative. The protein is encoded by the Ercc6l gene (Gene ID: 236930) and has been identified in the UniProt database with the primary accession number Q8BHK9 and entry name ERC6L_MOUSE . Unlike the better-characterized ERCC6 (also called CSB protein), which functions primarily in transcription-coupled excision repair, Ercc6l may have additional or variant functions in mouse cells that continue to be investigated by researchers.

What biological samples can be used for Ercc6l detection and analysis?

Ercc6l can be detected and analyzed using various biological materials from mice. The most common sample types include tissue homogenates (particularly from organs with high cell turnover rates), cell lysates from cultured mouse cell lines, and other biological fluids . For optimal results, samples should be processed according to standardized protocols that preserve protein integrity. Native samples generally provide more reliable results than recombinant proteins, as commercial detection methods are typically optimized for native protein detection. This optimization accounts for the natural folding patterns, post-translational modifications, and protein-protein interactions that may be altered in recombinant versions of the protein .

What are the primary differences between Ercc6l and ERCC6?

While related by name, Ercc6l (DNA excision repair protein ERCC-6-like) and ERCC6 (also known as CS-B protein) have distinct functions and properties. ERCC6 serves specifically as a transcription-coupled excision repair protein and is one of the fundamental enzymes in active gene repair . Mutations in ERCC6 are associated with Cockayne syndrome type II in humans. ERCC6 exhibits ATPase properties and contains conserved helicase motifs organized into two main domains that facilitate ATP binding and hydrolysis .

In contrast, Ercc6l appears to have broader functions beyond transcription-coupled repair. Research suggests that Ercc6l is involved in cellular processes related to chromosome segregation, DNA replication, and cell cycle regulation . The differential expression of Ercc6l in certain cancers, such as lung adenocarcinoma, also points to potential roles in cell proliferation pathways that may be distinct from the primarily repair-focused functions of ERCC6 .

What is the optimal approach for detecting Ercc6l protein expression in mouse samples?

For quantitative detection of Ercc6l in mouse samples, enzyme-linked immunosorbent assay (ELISA) represents one of the most reliable methodological approaches. Commercial ELISA kits for Ercc6l typically offer a detection range of 0.156 ng/ml to 10 ng/ml using colorimetric detection methods . For accurate results, sample concentrations should be diluted to mid-range of the detection spectrum.

The experimental protocol should include:

• Sample preparation: Proper homogenization of tissue or lysis of cells with protease inhibitors

• Standard curve preparation: Using lyophilized standards reconstituted according to manufacturer instructions

• Sample dilution optimization: Performing preliminary runs to identify appropriate dilution factors

• Controlled assay conditions: Maintaining consistent temperature, incubation times, and washing procedures

• Single-user processing: Ideally, the entire assay should be performed by the same researcher to minimize performance fluctuations

Alternative methods include western blotting for semi-quantitative analysis or immunohistochemistry for localization studies, though these may require additional validation steps for Ercc6l-specific applications.

How should researchers control for variability in Ercc6l detection assays?

Controlling for variability in Ercc6l detection assays requires careful attention to both experimental design and execution. The stability of detection kits is determined by the rate of activity loss, which should be less than 5% within the expiration date under appropriate storage conditions . To minimize performance fluctuations, researchers should implement the following controls:

• Technical replicates: Minimum of three per sample

• Biological replicates: Different animals or independently derived cell populations

• Standardized laboratory conditions: Strict control of temperature, humidity, and timing

• Consistent reagent handling: Using single lots of antibodies and detection reagents when possible

• Inclusion of reference samples: Known positive and negative controls in each experimental run

Additionally, it is strongly recommended that the entire assay procedure be performed by the same researcher throughout a study to minimize user-dependent variations . For long-term studies, creating aliquots of reference samples stored at -80°C can provide internal standards to normalize results across multiple experimental sessions.

What are the key considerations when working with recombinant versus native Ercc6l protein?

Working with recombinant Ercc6l presents distinct challenges compared to native protein analysis. Commercial detection kits are typically optimized for detecting native Ercc6l samples rather than recombinant proteins, making quantification of recombinant versions potentially less reliable .

Key considerations include:

• Structural differences: Recombinant proteins may have different tertiary structures compared to native proteins, affecting antibody binding and detection sensitivity

• Post-translational modifications: Native Ercc6l undergoes various modifications that may be absent in recombinant versions

• Partial versus full-length: Recombinant "partial" Ercc6l may lack domains present in the native protein

• Expression systems: The choice of bacterial, insect, or mammalian expression systems can significantly impact protein folding and function

• Validation requirements: Additional validation steps are necessary when using recombinant Ercc6l as a standard or control

Researchers should be aware that detection guarantees from commercial suppliers typically do not extend to recombinant proteins due to these differences . When working with recombinant Ercc6l, particularly partial constructs, additional characterization through methods such as mass spectrometry or circular dichroism may be necessary to confirm structural integrity before proceeding with functional studies.

How does Ercc6l expression correlate with disease states in experimental models?

In mouse models, altered Ercc6l expression might similarly correlate with cancer progression or other pathological states. Researchers investigating this connection should consider:

• Expression profiling across tissue types in healthy versus disease models

• Correlation analysis with established disease biomarkers

• Survival analysis in genetically modified mice with Ercc6l overexpression or knockdown

• Single-cell analysis to identify cell-specific expression patterns in heterogeneous tissues

The cancer single-cell state atlas (CancerSEA) has been used to investigate the function of cancer cells at the single-cell level, revealing significant differences in enrichment patterns . Similar approaches could be applied to mouse models to elucidate the role of Ercc6l in disease progression at cellular resolution.

What molecular pathways interact with Ercc6l based on co-expression analysis?

Functional enrichment analysis suggests that Ercc6l likely participates in several critical cellular processes:

Key pathways identified through KEGG analysis include cell cycle regulation, DNA replication, homologous recombination, oocyte meiosis, proteasome function, spliceosome activity, and p53 signaling . These findings suggest that Ercc6l functions within a complex network of proteins involved in maintaining genomic stability and regulating cell division, making it a potentially important factor in both normal development and disease states.

What are the structural and functional domains of Ercc6l that can be targeted in experimental studies?

Based on structural analysis of the related ERCC6 protein, researchers can make informed predictions about Ercc6l domains that might be important for experimental targeting. ERCC6 contains conserved helicase motifs organized into two main domains: domain 1 (comprising motifs I, Ia, II, and III) and domain 2 (comprising motifs IV, V, and VI) . These domains wrap around an interdomain cleft involved in ATP binding and hydrolysis.

For experimental studies targeting specific Ercc6l functions, researchers might consider:

• ATPase domain: Targeting the ATP binding and hydrolysis functions through site-directed mutagenesis of conserved residues in motifs I and II

• DNA binding regions: Investigating domain 2, which may affect DNA binding after ATP hydrolysis-induced conformational changes

• Protein interaction surfaces: Identifying regions involved in forming protein complexes at repair sites

• Regulatory regions: Exploring how post-translational modifications affect Ercc6l activity

How should researchers analyze Ercc6l expression data in relation to genetic and epigenetic factors?

Analysis of Ercc6l expression in relation to genetic and epigenetic factors requires integration of multiple data types. Evidence from human studies indicates that ERCC6L expression correlates positively with DNA copy number and negatively with DNA methylation , suggesting similar regulatory mechanisms may exist for mouse Ercc6l.

A comprehensive analytical approach should include:

• Correlation analysis between Ercc6l mRNA expression and copy number variations

• Assessment of promoter methylation status and its relationship to expression levels

• Investigation of potential transcription factors regulating Ercc6l expression

• Analysis of chromatin accessibility at the Ercc6l locus using techniques such as ATAC-seq

For statistical analysis, researchers should employ methods such as Pearson's correlation for continuous variables, t-tests or ANOVA for group comparisons, and multivariate regression to account for confounding factors. When analyzing publicly available datasets, care should be taken to account for batch effects and platform differences. For methylation studies, both gene body and promoter methylation should be considered, as their effects on expression can differ .

What approaches can resolve contradictory findings in Ercc6l functional studies?

Contradictory findings regarding Ercc6l function are not uncommon, particularly given the complexity of DNA repair mechanisms and potential redundancy within repair pathways. To address such contradictions, researchers should implement systematic approaches:

• Standardization of experimental conditions: Varying cell types, stress conditions, or assay methods can significantly impact results

• Dose-response analyses: Testing across concentration ranges can reveal threshold effects that might explain discrepancies

• Temporal considerations: Examining time-dependent effects through time-course experiments

• Context-dependent function analysis: Investigating how cellular context (cycling vs. quiescent cells, different tissues, etc.) affects Ercc6l activity

• Comparative analysis with related proteins: Studying the interplay between Ercc6l and other repair proteins

The contradictory publications regarding ATP concentration effects on the related CSB protein illustrate this challenge . Recent evidence suggests that ADP/AMP allosterically regulate CSB, indicating that ATP to ADP charge ratios may determine protein complex formation at repair sites . Similar allosteric regulation might explain contradictory findings in Ercc6l studies, highlighting the importance of considering nucleotide concentrations and ratios in experimental designs.

How can single-cell analysis advance our understanding of Ercc6l function in heterogeneous tissues?

Single-cell analysis represents a powerful approach to unravel Ercc6l functions in complex tissue environments. The cancer single-cell state atlas (CancerSEA) has been employed to investigate cellular functions at the single-cell level, revealing significant enrichment differences across cell populations . This approach can be particularly valuable for Ercc6l research given its apparent involvement in multiple cellular processes.

Key methodological considerations include:

• Single-cell RNA sequencing (scRNA-seq) to map Ercc6l expression across cell types

• Integration with spatial transcriptomics to understand tissue-context expression patterns

• Correlation of Ercc6l expression with functional cell states (proliferating, differentiating, senescent)

• Trajectory analysis to examine Ercc6l expression changes during developmental or disease progression

• Perturbation experiments at single-cell resolution to assess cell-type specific responses

Data analysis should employ the Spearman rank correlation test to analyze significant correlations between gene expression and functional status at the single-cell level, with appropriate correction for multiple comparisons through false discovery rate (FDR) adjustments . Dimensional reduction techniques such as t-SNE or UMAP can help visualize relationships between Ercc6l expression and cell states across heterogeneous populations.

What emerging technologies could advance Ercc6l research beyond current limitations?

Several cutting-edge technologies hold promise for overcoming current limitations in Ercc6l research:

• Cryo-electron microscopy (Cryo-EM): Could provide detailed structural information about Ercc6l protein complexes in native-like states

• Proximity labeling methods (BioID, APEX): Would help identify transient protein interactions that may be missed by traditional co-immunoprecipitation

• Live-cell imaging with fluorescent protein tags: Could track Ercc6l dynamics during DNA repair in real-time

• CRISPR-based epigenome editing: Would allow precise manipulation of Ercc6l regulation without altering the underlying DNA sequence

• Nanopore sequencing: Could identify DNA damage sites and repair intermediates with single-molecule resolution

These technologies would complement existing approaches and potentially resolve current contradictions in the literature. For instance, the disputed helicase activity of ERCC6 could be directly addressed through single-molecule biophysical techniques that monitor DNA unwinding in real-time.

How might comparative studies between mouse Ercc6l and human ERCC6L inform translational research?

Comparative studies between mouse Ercc6l and human ERCC6L represent a valuable approach for translational research. While similarities exist, there may also be important species-specific differences that affect experimental interpretation.

A comprehensive comparative analysis should include:

Such comparative approaches would help identify which aspects of mouse Ercc6l research are directly applicable to human health and disease. Given that ERCC6L overexpression correlates with poor prognosis in human lung adenocarcinoma , understanding the conservation of its cancer-related functions could inform the development of mouse models for cancer research and therapeutic development.