ERCC1 Human

Basic Research Questions

• What is ERCC1 and what is its primary function in human cells?

  ERCC1 is an essential component of the ERCC1-XPF endonuclease complex that participates in multiple DNA repair pathways. Its primary function involves forming a heterodimeric structure-specific endonuclease with XPF (also known as ERCC4 or FANCQ) that cleaves DNA 5' to damaged sites . This activity is crucial for nucleotide excision repair (NER), where the complex makes incisions on the damaged DNA strand, and for interstrand crosslink (ICL) repair, where it performs the "unhooking" step . ERCC1-XPF's endonuclease activity is fundamental for maintaining genomic integrity and preventing the accumulation of endogenous DNA damage.

• What DNA repair pathways involve ERCC1?

  ERCC1 participates in multiple DNA repair pathways:

  Repair Pathway	ERCC1 Role	Consequence of Deficiency
  Global Genome NER (GG-NER)	Makes 5' incision during repair	Photosensitivity, increased cancer risk
  Transcription-Coupled NER (TC-NER)	Makes 5' incision during repair	Photosensitivity, neurodegeneration
  Interstrand Crosslink Repair	DNA unhooking in Fanconi anemia pathway	Bone marrow failure, developmental abnormalities
  Homologous Recombination	Participates in certain sub-pathways	Genomic instability

  This involvement in multiple pathways explains why ERCC1 deficiency causes more severe phenotypes than defects in proteins involved in only one repair pathway .

• How does ERCC1 deficiency affect cellular function?

  ERCC1 deficiency profoundly impacts cellular function through multiple mechanisms:

  • Increased sensitivity to DNA-damaging agents, particularly UV radiation and crosslinking agents

  • Enhanced susceptibility to lipid peroxidation (LPO) products like 4-hydroxy-2-nonenal (HNE)

  • Accumulation of endogenous DNA damage leading to cellular senescence or apoptosis

  • Stimulation of reactive oxygen species (ROS) and further LPO formation, creating a damaging cycle

  • Induction of DNA base damage, strand breaks, and error-prone translesion DNA synthesis

  • Deregulation of base excision repair and energy production pathways

  • Inhibition of cellular proliferation

  These cellular dysfunctions collectively contribute to the severe progeroid phenotypes observed in ERCC1-deficient humans and mice.

Advanced Research Questions

• What are the known pathogenic mutations in the human ERCC1 gene and their phenotypic consequences?

  Several pathogenic ERCC1 mutations have been identified in humans:

  Mutation	Molecular Consequence	Clinical Phenotype	Reference
  Deletion + R156W	Protein instability, reduced DNA repair recruitment	Short stature, photosensitivity, severe liver and kidney impairment	Apelt et al., 2020
  Previously reported cases	Various molecular defects	Features of Cockayne syndrome, infantile death	Apelt et al., 2020

  The phenotypic spectrum of ERCC1 mutations includes features of multiple DNA repair disorders:

  • Xeroderma pigmentosum-like features: Photosensitivity

  • Cockayne syndrome-like features: Growth failure, neurodegeneration

  • Fanconi anemia-like features: Developmental abnormalities, bone marrow dysfunction

  The severity depends on the specific mutation's effect on ERCC1 protein stability and function.

• How do ERCC1 mutations specifically contribute to liver and kidney dysfunction?

  ERCC1 mutations lead to characteristic liver and kidney dysfunction through several mechanisms:

  Liver:

  • Accumulation of DNA damage in hepatocytes leads to cellular senescence

  • Increased sensitivity to endogenous metabolic byproducts

  • Enhanced susceptibility to lipid peroxidation products

  • Impaired liver regeneration and homeostasis

  Kidney:

  • Progressive accumulation of DNA damage in renal tubular cells

  • Impaired repair of damage caused by filtered toxins

  • Increased sensitivity to oxidative stress

  • Progressive loss of functional nephrons

  Both Apelt et al. and Garaycoechea et al. highlight that liver and kidney dysfunction are prominent features of ERCC1 deficiency in humans and mice . The extreme sensitivity of these organs to ERCC1 deficiency suggests their particular reliance on this DNA repair pathway for maintaining homeostasis.

• What is the relationship between ERCC1 deficiency and lipid peroxidation?

  ERCC1 deficiency and lipid peroxidation (LPO) interact in a complex relationship:

  • ERCC1-deficient cells and mice are hypersensitive to LPO products including 4-hydroxy-2-nonenal (HNE), crotonaldehyde, and malondialdehyde

  • LPO products induce DNA damage including crosslinks that require ERCC1-XPF for repair

  • When exposed to HNE, ERCC1-deficient cells show:

    • Greater inhibition of proliferation

    • Increased ROS production

    • Enhanced DNA damage

    • Accelerated cellular senescence

  • ERCC1-deficient mice show increased sensitivity to CCl4 (a LPO inducer) and diets rich in polyunsaturated fatty acids

  • LPO products can inhibit DNA repair pathways, compounding the repair defect

  This relationship suggests dietary interventions targeting LPO might benefit patients with ERCC1 deficiency, and that accumulation of LPO products may contribute to aging-related pathologies even in individuals with normal ERCC1 function .

• How does ERCC1 deficiency affect hematopoietic stem cell function?

  ERCC1 deficiency severely impacts hematopoietic stem cell (HSC) function:

  • Ercc1-/- mice exhibit a profound reduction in HSC frequency compared to wild-type mice

  • The HSC defect in Ercc1-/- mice (11.8-fold reduction) is significantly more severe than in Fanca-/- mice (1.8-fold reduction)

  • This defect begins during embryonic development (by E13.5), preceding liver and kidney dysfunction

  • Xpa-/- mice, deficient only in NER, do not show significant HSC reduction, suggesting ERCC1's role extends beyond NER

  • The severe HSC defect likely contributes to hematopoietic abnormalities observed in patients with ERCC1 mutations

  Garaycoechea et al. demonstrated that "ERCC1 deficiency removes not only the dominant FA ICL-repair pathway, but also an additional pathway of HSC protection" . This finding highlights ERCC1's multifunctional role in maintaining hematopoietic homeostasis.

• What experimental models are best suited for studying ERCC1 deficiency?

  Several experimental models are available for studying ERCC1 deficiency:

  Cellular Models:

  • Primary mouse embryonic fibroblasts (MEFs) from Ercc1-/- mice

  • SV40-immortalized MEFs (wild-type and Ercc1-/-)

  • Xpa-/- MEFs (for comparison with NER-only deficiency)

  • CRISPR/Cas9-engineered human cell lines (e.g., HAP1)

  Mouse Models:

  • Ercc1-/- (complete knockout): Severe phenotype with ~4 week lifespan

  • Ercc1-/Δ (hypomorphic): ~5% normal ERCC1-XPF expression, ~7 month lifespan

  • Fanca-/- mice (for comparison with ICL-repair deficiency)

  The choice of model depends on the research question:

  • Complete knockout models are suitable for studying developmental effects

  • Hypomorphic models allow for studying progressive disease

  • Comparison with pathway-specific models (Xpa-/-, Fanca-/-) helps delineate ERCC1's distinct functions

  When designing experiments, researchers should consider the limitations of each model and the specific aspects of ERCC1 function they aim to investigate.

• How does ERCC1 function outside canonical excision repair pathways?

  ERCC1 has several functions outside its well-characterized roles in NER and ICL repair:

  • Protection of liver and kidney homeostasis through mechanisms independent of canonical repair pathways

  • Maintenance of hematopoietic stem cell populations through pathways distinct from both NER and FA repair

  • Response to oxidative stress and lipid peroxidation products

  • Protection against endogenous DNA damage sources

  Garaycoechea et al. explicitly state that "XPF-ERCC1 has important functions outside of its central role in NER and FA crosslink repair which are required to prevent endogenous DNA damage" . These non-canonical functions likely contribute to the severe phenotype observed in ERCC1-deficient organisms, which cannot be fully explained by defects in NER and ICL repair alone.

• What are the cellular responses to ERCC1 deficiency in experimental models?

  ERCC1-deficient cells exhibit several characteristic responses:

  • Hypersensitivity to DNA damaging agents, particularly crosslinking agents and UV radiation

  • Enhanced susceptibility to lipid peroxidation products like HNE

  • Inhibition of proliferation following DNA damage exposure

  • Increased ROS and continued LPO formation creating a damaging cycle

  • Induction of cellular senescence

  • Diploidization in haploid cell models (observed in XPF-deficient HAP1 cells)

  • Altered cell cycle progression

  • Transcriptional changes affecting multiple cellular pathways

  These cellular responses provide insights into the mechanisms underlying the tissue-specific pathologies observed in ERCC1-deficient organisms and potential therapeutic targets for intervention.

Methodological Approaches

• How can ERCC1 function be assessed in patient-derived samples?

  Assessment of ERCC1 function can be performed using several methodologies:

  Protein Analysis:

  • Western blotting to detect ERCC1 protein levels

  • Immunoprecipitation to assess ERCC1-XPF complex formation

  Functional Assays:

  • Sensitivity testing to DNA-damaging agents (UV, cisplatin, mitomycin C)

  • Cell viability assessments (e.g., alamarBlue assay)

  • Proliferation kinetics after DNA damage exposure

  Cellular Phenotyping:

  • DNA damage markers (e.g., γH2AX foci)

  • Senescence markers (e.g., SA-β-galactosidase)

  • ROS measurement

  When interpreting results, consider:

  • The specific mutation and its predicted effect on ERCC1 function

  • Cell type being analyzed

  • Passage number of cultured cells

  A comprehensive assessment using multiple approaches provides the most reliable evaluation of ERCC1 function in patient samples.

• What strategies can be employed to study the role of ERCC1 in specific tissues?

  Several strategies can be employed to study tissue-specific ERCC1 functions:

  • Animal models with tissue-specific ERCC1 knockout or hypomorphic expression

  • Comparative analysis between tissues in systemic ERCC1-deficient models to identify differential sensitivity

  • Ex vivo culture of specific tissues from ERCC1-deficient animals

  • Induced pluripotent stem cell (iPSC) models derived from patients with ERCC1 mutations, differentiated into specific cell types

  • Tissue-specific biomarkers of DNA damage and repair in ERCC1-deficient models

  • Transcriptomic and proteomic profiling of different tissues from ERCC1-deficient organisms

  For example, Garaycoechea et al. performed detailed quantitative analysis of hematopoietic stem cell populations from Ercc1-/- mice compared to other DNA repair-deficient models to elucidate ERCC1's specific role in hematopoiesis .

• How can researchers differentiate the impact of different DNA repair deficiencies in ERCC1-deficient models?

  Differentiating the contributions of various repair pathways in ERCC1-deficient models requires:

  • Comparison with models deficient in only one pathway (e.g., Xpa-/- for NER, Fanca-/- for ICL repair)

  • Epistasis analysis by generating double or triple mutants

  • Complementation studies using mutant forms of ERCC1 with selective deficiencies

  • Pathway-specific DNA damage induction:

    • UV irradiation for NER

    • Crosslinking agents for ICL repair

    • Lipid peroxidation inducers for non-canonical functions

  • Cell type-specific analysis where certain repair pathways predominate

  Garaycoechea et al. demonstrate this approach by comparing phenotypes across multiple DNA repair-deficient mouse models, concluding that "joint inactivation of GG-NER, TC-NER and FA crosslink repair cannot account for the hypersensitivity of XPF-deficient cells to classical crosslinking agents nor is it sufficient to explain the extreme phenotype of Ercc1-/- mice" .

• What techniques are effective for studying ERCC1's role in preventing premature aging?

  To study ERCC1's role in preventing premature aging, researchers can employ:

  • Lifespan and healthspan analysis of ERCC1-deficient models

  • Molecular markers of aging (e.g., senescence-associated β-galactosidase, p16INK4a expression)

  • Tissue histopathology focused on age-related changes

  • Comparative transcriptomics between ERCC1-deficient tissues and naturally aged tissues

  • Interventional studies testing anti-aging compounds in ERCC1-deficient models

  • Dietary interventions, particularly those targeting lipid peroxidation

  • Longitudinal assessment of organ function (liver, kidney, hematopoietic system)

  • Biomarkers of DNA damage accumulation over time

  For example, research showing that ERCC1-deficient mice are hypersensitive to lipid peroxidation suggests that "LPO-induced DNA damage contributes to cellular demise and tissue degeneration" and may be a targetable mechanism in premature aging .

• What experimental approaches can elucidate the mechanisms of liver and kidney dysfunction in ERCC1 deficiency?

  To investigate liver and kidney dysfunction mechanisms in ERCC1 deficiency:

  Liver:

  • Histopathological analysis of liver sections

  • Liver function tests (ALT, AST, bilirubin)

  • Assessment of lipid peroxidation levels in liver tissue

  • Transcriptomic analysis of hepatic gene expression

  • Isolation and culture of primary hepatocytes from ERCC1-deficient models

  • CCl4 challenge studies to assess sensitivity to induced liver damage

  Kidney:

  • Histopathological analysis of kidney sections

  • Renal function tests (creatinine, BUN)

  • Analysis of proteinuria and microalbuminuria

  • Immunohistochemical detection of DNA damage markers in renal tubular cells

  • Assessment of renal response to nephrotoxic compounds

  Both tissues:

  • In vivo imaging to assess functional changes

  • Electron microscopy to detect ultrastructural changes

  • Single-cell sequencing to identify particularly affected cell populations

  The search results indicate that both liver and kidney dysfunction are prominent features of ERCC1 deficiency and likely contribute significantly to the reduced lifespan observed in ERCC1-deficient organisms .