Numerous proteins are involved in the multiple pathways of the DNA damage response network and play a key role to protect the genome from the wide variety of damages that can occur to DNA. An example of this is the structure-specific endonuclease ERCC1-XPF. This heterodimeric complex is in particular involved in nucleotide excision repair (NER), but also in double strand break repair and interstrand cross-link repair pathways. Here we review the function of ERCC1-XPF in various DNA repair pathways and discuss human disorders associated with ERCC1-XPF deficiency. We also overview our molecular and structural understanding of XPF-ERCC1.

Pathogenic variants in genes, which encode DNA repair and damage response proteins, result in a number of genomic instability syndromes with features of accelerated aging. ERCC4 (XPF) encodes a protein that forms a complex with ERCC1 and is required for the 5' incision during nucleotide excision repair. ERCC4 is also FANCQ, illustrating a critical role in interstrand crosslink repair. Pathogenic variants in this gene cause xeroderma pigmentosum, XFE progeroid syndrome, Cockayne syndrome (CS), and Fanconi anemia. We performed massive parallel sequencing for 42 unsolved cases submitted to the International Registry of Werner Syndrome. Two cases, each carrying two novel heterozygous ERCC4 variants, were identified. The first case was a compound heterozygote for: c.2395C > T (p.Arg799Trp) and c.388+1164_792+795del (p.Gly130Aspfs*18). Further molecular and cellular studies indicated that the ERCC4 variants in this patient are responsible for a phenotype consistent with a variant of CS. The second case was heterozygous for two variants in cis: c.[1488A > T; c.2579C > A] (p.[Gln496His; Ala860Asp]). While the second case also had several phenotypic features of accelerated aging, we were unable to provide biological evidence supporting the pathogenic roles of the associated ERCC4 variants. Precise genetic causes and disease mechanism of the second case remains to be determined.

Background: An F231L mutation in the DNA repair complex ERCC1-XPF causes severe disorders. Results: The observed enhanced dissociation of the mutant ERCC1-XPF helix-hairpin-helix dimer relates to an altered orientation for the interaction anchor Phe 894 of XPF near Phe 231 of ERCC1. Conclusion: The local structure around Phe 231 is essential for complex stability. Significance: Studying mutations interfering with DNA repair is crucial to understand endonuclease-related diseases.

Edited by Patrick SungThe nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the helix-hairpin-helix (HhH) domains of XPF and ERCC-XPF and show that the binding to single-stranded DNA (ssDNA)/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences but with a clear preference for guanine-containing substrates. NMR titration experiments and in vitro DNA binding assays also show that, within the heterodimeric ERCC1-XPF complex, XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-overlapping DNA binding domains in the ERCC1-XPF heterodimer jointly bind to an ssDNA/dsDNA substrate and, thereby, at least partially dictate the incision position during damage removal. Based on structural models, NMR titrations, DNA-binding studies, site-directed mutagenesis, charge distribution, and sequence conservation, we propose that the HhH domain of ERCC1 binds to dsDNA upstream of the damage, and XPF binds to the non-damaged strand within a repair bubble.To survive, cells require the ability to repair a plethora of DNA lesions. Therefore, cells contain several DNA repair mechanisms, including the versatile nucleotide excision repair (NER) 2 pathway, a conserved DNA repair machinery that can remove a wide variety of DNA lesions (1, 2). Within a mammalian cell, 25-30 proteins are known to participate in two NER pathways: global genome and transcription coupled repair (3)(4)(5). Mutations in NER genes lead to impaired DNA repair. Presently, a dozen mutations in distinct NER genes have been identified in patients with eight overlapping phenotypes (6, 7). Most patients carrying a mutation in NER genes develop two distinct symptoms: sunlight-induced skin cancer and segmental progeria without cancer (8, 9). ERCC1 and XPF form a stable heterodimeric complex that is essential for NER and functions as a structure-specific DNA endonuclease that is able to perform an incision 5Ј to the DNA damage (10 -13). Mutations in the ERCC1 and XPF genes can be linked to sunlight-induced skin abnormalities, late onset of skin cancers, neurodegeneration, and premature aging in both human patients and mice (7)(8)(9)14). In the absence of ERCC1, only a marginal amount of XPF is present in fibroblasts and CHO cells (11,13,(15)(16)(17)(18). This suggests that the in vivo stability of full-length ERCC1-XPF depends on tight association between the two proteins. Consistent with this finding, XPF and ERCC1 knockout mice exhibit similar phenotypes (19 -21). Furthermore, postnatal phenotypes of XPF and E...

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Human prion diseases are associated with misfolding or aggregation of the Human Prion Protein (HuPrP). Missense mutations in the HuPrP gene, contribute to conversion of HuPrP(C) to HuPrP(Sc) and amyloid formation. Based on our previous comprehensive study, three missense mutations, from two different functional groups, i.e. disease-related mutations, and protective mutations, were selected and extensive molecular dynamics simulations were performed on these three mutants to compare their dynamics and conformations with those of the wildtype HuPrP. In addition to simulations of monomeric forms of mutants, in order to study the dominant-negative effect of protective mutation (E219K), 30-ns simulations were performed on E219K-wildtype and wildtype-wildtype dimeric forms. Our results indicate that, although after 30-ns simulations the global three-dimensional structure of models remain fairly intact, the disease-related mutations (V210I and Q212P) introduce local structural changes, i.e. close contact changes and secondary structure changes, in addition to global flexibility changes. Furthermore, our results support the loss of hydrophobic interaction due to the mutations in hydrophobic core that has been reported by previous NMR and computational studies. On the other hand, this protective mutation (E219K) results in helix elongation, and significant increases of overall flexibility of E219K mutant during 30-ns simulation. In conclusion, the simulations of dimeric forms suggest that the dominant-negative effect of this protective mutation (E219K) is due to the incompatible structures and dynamics of allelic variants during conversion process.

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Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by KLHL16 mutations. KLHL16 encodes gigaxonin, a regulator of intermediate filament (IF) protein turnover. Previous neuropathological studies and our own examination of postmortem GAN brain tissue in the current study revealed significant involvement of astrocytes in GAN. To study the underlying cellular mechanisms, we generated human models of GAN using induced pluripotent stem cells (iPSCs). Skin fibroblasts from seven GAN patients carrying different KLHL16 mutations were reprogrammed to iPSCs, and isogenic controls were derived via CRISPR/Cas9 editing. Neural progenitor cells (NPCs), astrocytes, and brain organoids were generated through directed differentiation. All GAN iPSC lines were deficient for gigaxonin, which was restored in the isogenic clones. While GAN iPSCs displayed normal organization of lamin B1 and keratin IFs, they exhibited patient-specific increased expression and perinuclear bundling of vimentin. Nestin IF morphology was unaffected, but fewer nestin-positive cells were present in GAN NPCs compared to controls. The most dramatic phenotypes were observed in GAN iPSC-astrocytes and brain organoids, which displayed dense perinuclear IF accumulations and abnormal nuclear morphology. GFAP oligomerization and perinuclear aggregation were strongly potentiated in the presence of vimentin, and GAN cells with large perinuclear vimentin aggregates accumulated nuclear KLHL16 mRNA. As an early effector of KLHL16 mutations, vimentin may be a potential target in GAN.