ERCC4

ERCC4 is a is_associated_with::protein designated as DNA repair endonuclease XPF that in humans is encoded by the ERCC4 is_associated_with::gene. Together with is_associated_with::ERCC1, ERCC4 forms the ERCC1-XPF enzyme complex that participates in is_associated_with::DNA repair and is_associated_with::DNA recombination.

The is_associated_with::nuclease enzyme ERCC1-XPF cuts specific structures of DNA. Many aspects of these two gene products are described together here because they are partners during DNA repair. The ERCC1-XPF nuclease is an essential activity in the pathway of DNA is_associated_with::nucleotide excision repair (NER). The ERCC1-XPF nuclease also functions in pathways to repair is_associated_with::double-strand breaks in DNA, and in the repair of “crosslink” damage that harmfully links the two DNA strands.

Cells with disabling mutations in ERCC4 are more sensitive than normal to particular DNA damaging agents, including is_associated_with::ultraviolet radiation and to chemicals that cause crosslinking between DNA strands. Genetically engineered mice with disabling mutations in ERCC4 also have defects in DNA repair, accompanied by metabolic stress-induced changes in physiology that result in premature aging. Complete deletion of ERCC4 is incompatible with viability of mice, and no human individuals have been found with complete (homozygous) deletion of ERCC4. Rare individuals in the human population harbor inherited mutations that impair the function of ERCC4. When the normal genes are absent, these mutations can lead to human syndromes, including is_associated_with::xeroderma pigmentosum, is_associated_with::Cockayne syndrome and is_associated_with::Fanconi anemia.

ERCC1 and ERCC4 are the human gene names and Ercc1 and Ercc4 are the analogous mammalian gene names. Similar genes with similar functions are found in all eukaryotic organisms.

Gene
The human ERCC4 gene can correct the DNA repair defect in specific ultraviolet light (UV)-sensitive mutant cell lines derived from Chinese hamster ovary cells. Multiple independent complementation groups of Chinese hamster ovary (CHO) cells have been isolated, and this gene restored UV resistance to cells of complementation group 4. Reflecting this cross-species genetic complementation method, the gene was called “Excision repair cross-complementing 4”

The human ERCC4 gene encodes the XPF protein of 916 amino acids with a molecular mass of about 104,000 daltons.

Genes similar to ERCC4 with equivalent functions (orthologs) are found in other eukaryotic genomes. Some of the most studied gene orthologs include RAD1 in the budding yeast Saccharomyces cerevisiae, and rad16+ in the fission yeast Schizosaccharomyces pombe.

Protein


One ERCC1 molecule and one XPF molecule bind together, forming an ERCC1-XPF heterodimer which is the active nuclease form of the enzyme. In the ERCC1–XPF heterodimer, ERCC1 mediates DNA– and protein–protein interactions. XPF provides the endonuclease active site and is involved in DNA binding and additional protein–protein interactions.

The ERCC4/XPF protein consists of two conserved areas separated by a less conserved region in the middle. The N-terminal area has homology to several conserved domains of DNA helicases belonging to superfamily II, although XPF is not a DNA helicase. The C-terminal region of XPF includes the active site residues for nuclease activity. (Figure 1).

Most of the ERCC1 protein is related at the sequence level to the C terminus of the XPF protein., but residues in the nuclease domain are not present. A DNA binding “helix-hairpin-helix” domain at the C-terminus of each protein.

By primary sequence and protein structural similarity, the ERCC1-XPF nuclease is a member of a broader family of structure specific DNA nucleases comprising two subunits. Such nucleases include, for example, the MUS81-EME1 nuclease.

Structure-specific nuclease


The ERCC1–XPF complex is a structure-specific endonuclease. ERCC1-XPF does not cut DNA that is exclusively single-stranded or double-stranded, but it cleaves the DNA phosphodiester backbone specifically at junctions between double-stranded and single-stranded DNA. It introduces a cut in double-stranded DNA on the 5′ side of such a junction, about two nucleotides away (Figure 2). This structure-specificity was initially demonstrated for RAD10-RAD1, the yeast orthologs of ERCC1 and XPF.

The hydrophobic helix–hairpin–helix motifs in the C-terminal regions of ERCC1 and XPF interact to promote dimerization of the two proteins. There is no catalytic activity in the absence of dimerization. Indeed, although the catalytic domain is within XPF and ERCC1 is catalytically inactive, ERCC1 is indispensable for activity of the complex.

Several models have been proposed for binding of ERCC1–XPF to DNA, based on partial structures of relevant protein fragments at atomic resolution. DNA binding mediated by the helix-hairpin-helix domains of ERCC1 and XPF domains positions the heterodimer at the junction between double-stranded and single-stranded DNA.

Nucleotide excision repair (NER)
During nucleotide excision repair, several protein complexes cooperate to recognize damaged DNA and locally separate the DNA helix for a short distance on either side of the site of a site of DNA damage. The ERCC1–XPF nuclease incises the damaged DNA strand on the 5′ side of the lesion. During NER, the ERCC1 protein interacts with the XPA protein to coordinate DNA and protein binding.

DNA double-strand break (DSB) repair
Mammalian cells with mutant ERCC1–XPF are moderately more sensitive than normal cells to agents (such as ionizing radiation) that cause double-stranded breaks in DNA. Particular pathways of both homologous recombination repair and non-homologous end-joining rely on ERCC1-XPF function. The relevant activity of ERCC1–XPF for both types of double-strand break repair is the ability to remove non-homologous 3′ single-stranded tails from DNA ends before rejoining. This activity is needed during a single-strand annealing subpathway of homologous recombination. Trimming of 3’ single-stranded tails is also needed in a mechanistically distinct subpathway of non-homologous end-joining, independent of the Ku proteins Homologous integration of DNA, an important technique for genetic manipulation, is dependent on the function of ERCC1-XPF in the host cell.

Interstrand crosslinks repair
Mammalian cells carrying mutations in ERCC1 or XPF are especially sensitive to agents that cause DNA interstrand crosslinks (ICL) Interstrand crosslinks block the progression of DNA replication, and structures at blocked DNA replication forks provide substrates for cleavage by ERCC1-XPF. Incisions may be made on either side of the crosslink on one DNA strand to unhook the crosslink and initiate repair. Alternatively, a double-strand break may be made in the DNA near the ICL, and subsequent homologous recombination repair my involve ERCC1-XPF action. Although not the only nuclease involved, ERCC1–XPF is required for ICL repair during several phases of the cell cycle.

Xeroderma pigmentosum (XP)
Some individuals with the rare inherited syndrome is_associated_with::xeroderma pigmentosum have mutations in ERCC4. These patients are classified as XP complementation group F (XP-F). Diagnostic features of XP are dry scaly skin, abnormal skin pigmentation in sun-exposed areas and severe photosensitivity, accompanied by a great than 1000-fold increased risk of developing UV radiation-induced skin cancers.

Cockayne syndrome (CS)
Most XP-F patients show moderate symptoms of XP, but a few show additional symptoms of Cockayne syndrome. Cockayne syndrome (CS) patients exhibit photosensitivity, and also exhibit developmental defects and neurological symptoms.

Mutations in the ERCC4 gene can result in the very rare XF-E syndrome. These patients have characteristics of XP and CS, as well as additional neurologic, hepatobiliary, musculoskeletal and hematopoietic symptoms.

Fanconi anemia
Several human patients with symptoms of Fanconi anemia (FA) have causative mutations in the ERCC4 gene. Fanconi anemia is a complex disease, involving major hematopoietic symptoms. A characteristic feature of FA is the hypersensitivity to agents that cause interstrand DNA crosslinks. FA patients with ERCC4 mutations have been classified as belonging to Fanconi anemia complementation group P (FANCP).