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ERCC4 is a protein designated as DNA repair endonuclease XPF that in humans is encoded by the ''ERCC4''
gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ...
. Together with
ERCC1 DNA excision repair protein ERCC-1 is a protein that in humans is encoded by the ''ERCC1'' gene. Together with ERCC4, ERCC1 forms the ERCC1-XPF enzyme complex that participates in DNA repair and DNA recombination. Many aspects of these two gen ...
, ERCC4 forms the ERCC1-XPF enzyme complex that participates in
DNA repair DNA repair is a collection of processes by which a cell (biology), cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolism, metabolic activities and environmental factors such as r ...
and DNA recombination. The
nuclease A nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their ...
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 nucleotide excision repair (NER). The ERCC1-XPF nuclease also functions in pathways to repair
double-strand break DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA dama ...
s 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
ultraviolet radiation Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nm (with a corresponding frequency around 30  PHz) to 400 nm (750  THz), shorter than that of visible light, but longer than X-rays. UV radiatio ...
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 xeroderma pigmentosum,
Cockayne syndrome Cockayne syndrome (CS), also called Neill-Dingwall syndrome, is a rare and fatal autosomal recessive neurodegenerative disorder characterized by growth failure, impaired development of the nervous system, abnormal sensitivity to sunlight ( photo ...
and
Fanconi anemia Fanconi anaemia (FA) is a rare genetic disease resulting in impaired response to DNA damage. Although it is a very rare disorder, study of this and other bone marrow failure syndromes has improved scientific understanding of the mechanisms of nor ...
. ''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.


Clinical significance


Xeroderma pigmentosum (XP)

Some individuals with the rare inherited syndrome 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 Q (FANCQ).


ERCC4 (XPF) in the normal colon

ERCC4 (XPF) is normally expressed at a high level in cell nuclei within the inner surface of the colon (see image, panel C). The inner surface of the colon is lined with simple columnar
epithelium Epithelium or epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. It is a thin, continuous, protective layer of compactly packed cells with a little intercel ...
with invaginations. The invaginations are called
intestinal gland In histology, an intestinal gland (also crypt of Lieberkühn and intestinal crypt) is a gland found in between villi in the intestinal epithelium lining of the small intestine and large intestine (or colon). The glands and intestinal villi are c ...
s or colon crypts. The colon crypts are shaped like microscopic thick walled test tubes with a central hole down the length of the tube (the crypt lumen). Crypts are about 75 to 110 cells long. DNA repair, involving high expression of ERCC4 (XPF), PMS2 and ERCC1 proteins, appears to be very active in colon crypts in normal, non-
neoplastic A neoplasm () is a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists ...
colon epithelium. Cells are produced at the crypt base and migrate upward along the crypt axis before being shed into the colonic lumen days later. There are 5 to 6
stem cell In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type of ...
s at the bases of the crypts. There are about 10 million crypts along the inner surface of the average human colon. If the
stem cell In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type of ...
s at the base of the crypt express ERCC4 (XPF), generally all several thousand cells of the crypt will also express ERCC4 (XPF). This is indicated by the brown color seen by immunostaining of ERCC4 (XPF) in almost all the cells in the crypt in panel C of the image in this section. A similar expression of PMS2 and ERCC1 occurs in the thousands of cells in each normal colonic crypt. The tissue section in the image shown here was also
counterstain A counterstain is a stain with colour contrasting to the principal stain, making the stained structure easily visible using a microscope. Examples include the malachite green counterstain to the fuchsine stain in the Gimenez staining technique ...
ed with
hematoxylin Haematoxylin or hematoxylin (), also called natural black 1 or C.I. 75290, is a compound extracted from heartwood Wood is a porous and fibrous structural tissue found in the stems and roots of trees and other woody plants. It is an o ...
to stain DNA in nuclei a blue-gray color. Nuclei of cells in the
lamina propria The lamina propria is a thin layer of connective tissue that forms part of the moist linings known as mucous membranes or mucosae, which line various tubes in the body, such as the respiratory tract, the gastrointestinal tract, and the urogenit ...
, cells which are below and surround the epithelial crypts, largely show hematoxylin blue-gray color and have little expression of PMS2, ERCC1 or ERCC4 (XPF). In addition, cells at the very tops of the crypts stained for PMS2 (panel A) or ERCC4 (XPF) (panel C) have low levels of these DNA repair proteins, so that such cells show the blue-gray DNA stain as well.


ERCC4 (XPF) deficiency in the colon epithelium adjacent to and within cancers

ERCC4 (XPF) is deficient in about 55% of colon cancers, and in about 40% of the colon crypts in the epithelium within 10 cm adjacent to the cancers (in the field defects from which the cancers likely arose). When ERCC4 (XPF) is reduced in colonic crypts in a field defect, it is most often associated with reduced expression of DNA repair enzymes ERCC1 and PMS2 as well, as illustrated in the image in this section. Deficiencies in ERCC1 (XPF) in colon epithelium appear to be due to
epigenetic In biology, epigenetics is the study of stable phenotypic changes (known as ''marks'') that do not involve alterations in the DNA sequence. The Greek prefix '' epi-'' ( "over, outside of, around") in ''epigenetics'' implies features that are "o ...
repression. A deficiency of ERCC4 (XPF) would lead to reduced repair of DNA damages. As indicated by Harper and Elledge, defects in the ability to properly respond to and repair DNA damage underlie many forms of cancer. The frequent epigenetic reduction in ERCC4 (XPF) in field defects surrounding colon cancers as well as in cancers (along with epigenetic reductions in ERCC1 and PMS2) indicate that such reductions may often play a central role in progression to colon cancer. Although epigenetic reductions in ERCC4 (XPF) expression are frequent in human colon cancers, mutations in ERCC4 (XPF) are rare in humans. However, a mutation in ERCC4 (XPF) causes patients to be prone to skin cancer. An inherited polymorphism in ERCC4 (XPF) appears to be important in breast cancer as well. These infrequent mutational alterations underscore the likely role of ERCC4 (XPF) deficiency in progression to cancer.


Notes


References


External links


GeneReviews/NIH/NCBI/UW entry on xeroderma pigmentosum
{{DNA repair Human proteins