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109-507: 1Z00 , 2A1I , 2A1J , 2JNW , 2JPD , 2MUT 2067 13870 ENSG00000012061 ENSMUSG00000003549 P07992 P07903 NM_001166049 NM_001983 NM_202001 NM_001127324 NM_007948 NP_001356339 NP_001356340 NP_001356341 NP_001356342 NP_001356343 NP_001356344 NP_001356345 NP_001356346 NP_001356347 NP_001356348 NP_001120796 NP_031974 DNA excision repair protein ERCC-1

218-520: A carboxyl group, and a variable side chain are bonded . Only proline differs from this basic structure as it contains an unusual ring to the N-end amine group, which forces the CO–NH amide moiety into a fixed conformation. The side chains of the standard amino acids, detailed in the list of standard amino acids , have a great variety of chemical structures and properties; it is the combined effect of all of

327-470: A gene may be duplicated before it can mutate freely. However, this can also lead to complete loss of gene function and thus pseudo-genes . More commonly, single amino acid changes have limited consequences although some can change protein function substantially, especially in enzymes . For instance, many enzymes can change their substrate specificity by one or a few mutations. Changes in substrate specificity are facilitated by substrate promiscuity , i.e.

436-527: A recessive one that codes for a malfunctioning protein ( a and b respectively). Since both proteins are necessary for the synthesis of red pigmentation in the eyes, if a given fly is homozygous for either a or b , it will have white eyes. In genetics, a complementation test can be conducted to understand the interaction between different genetic strains. This test often involves crossing two pure-breeding strains, such as white-eyed flies, from separate origins. The process entails mating two flies, each from

545-478: A F231L mutation. Measuring ERCC1 activity may have utility in clinical cancer medicine because one mechanism of resistance to platinum chemotherapy drugs correlates with high ERCC1 activity. Nucleotide excision repair (NER) is the primary DNA repair mechanism that removes the therapeutic platinum-DNA adducts from the tumor DNA. ERCC1 activity levels, being an important part of the NER common final pathway, may serve as

654-552: A combination of sequence, structure and function, and they can be combined in many different ways. In an early study of 170,000 proteins, about two-thirds were assigned at least one domain, with larger proteins containing more domains (e.g. proteins larger than 600 amino acids having an average of more than 5 domains). Most proteins consist of linear polymers built from series of up to 20 different L -α- amino acids. All proteinogenic amino acids possess common structural features, including an α-carbon to which an amino group,

763-403: A defined conformation . Proteins can interact with many types of molecules, including with other proteins , with lipids , with carbohydrates , and with DNA . It has been estimated that average-sized bacteria contain about 2 million proteins per cell (e.g. E. coli and Staphylococcus aureus ). Smaller bacteria, such as Mycoplasma or spirochetes contain fewer molecules, on

872-851: A detailed review of the vegetable proteins at the Connecticut Agricultural Experiment Station . Then, working with Lafayette Mendel and applying Liebig's law of the minimum , which states that growth is limited by the scarcest resource, to the feeding of laboratory rats, the nutritionally essential amino acids were established. The work was continued and communicated by William Cumming Rose . The difficulty in purifying proteins in large quantities made them very difficult for early protein biochemists to study. Hence, early studies focused on proteins that could be purified in large quantities, including those of blood, egg whites, and various toxins, as well as digestive and metabolic enzymes obtained from slaughterhouses. In

981-402: A different strain. The eye color of the resulting progeny determines the outcome of the test. If the progeny exhibit red eyes, it indicates that the two strains complement each other. Conversely, if the progeny have white eyes, it suggests non-complementation. Complementation occurs when each strain possesses a different homozygous mutation (for example, one strain having the genotype 'aa BB' and

1090-519: A high risk of cancer (see inherited impairment in DNA repair increases cancer risk ), somatic mutations in DNA repair genes, including ERCC1 , only occur at low levels in sporadic (non-familial) cancers. Control of ERCC1 protein level occurred at the translational level. In addition to the wild-type sequence, three splice variants of mRNA ERCC1 exist. ERCC1 mRNA is also found to have either wild-type or three alternative transcription start points. Neither

1199-434: A key Phenylalanine residue from XPF (F894) and the mutation (F231L) disturbs this accommodating function. As a consequence, F894 protrudes out of the interface and the mutant complex is dissociating faster compared to the native one. The life span of patients with such mutations is often around 1–2 years. One Cockayne syndrome (CS) type II patient designated CS20LO exhibited a homozygous mutation in exon 7 of ERCC1, producing

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1308-478: A limited lifespan. Accelerated aging in the mutant involves various organs. Ercc1 mutant mice are deficient in several DNA repair processes including transcription -coupled DNA repair. This deficiency prevents resumption of RNA synthesis on the template DNA strand subsequent to it receiving a transcription-blocking DNA damage . Such blockages of transcription appear to promote premature aging, particularly in non-proliferating or slowly proliferating organs such as

1417-478: A little ambiguous and can overlap in meaning. Protein is generally used to refer to the complete biological molecule in a stable conformation , whereas peptide is generally reserved for a short amino acid oligomers often lacking a stable 3D structure. But the boundary between the two is not well defined and usually lies near 20–30 residues. Polypeptide can refer to any single linear chain of amino acids, usually regardless of length, but often implies an absence of

1526-648: A marker of general NER throughput. This has been suggested for patients with gastric, ovarian and bladder cancers. In Non-small cell lung carcinoma (NSCLC), surgically removed tumors that receive no further therapy have a better survival if ERCC1-positive than if ERCC1-negative. Thus, ERCC1 positivity is a favorable prognostic marker, referring to how the disease will proceed if not further treated. ERCC1-positive NSCLC tumors do not benefit from adjuvant platinum chemotherapy. However, ERCC1-negative NSCLC tumors, prognostically worse without treatment, derive substantial benefit from adjuvant cisplatin-based chemotherapy. High ERCC1

1635-410: A particular cell or cell type is known as its proteome . The chief characteristic of proteins that also allows their diverse set of functions is their ability to bind other molecules specifically and tightly. The region of the protein responsible for binding another molecule is known as the binding site and is often a depression or "pocket" on the molecular surface. This binding ability is mediated by

1744-500: A protein carries out its function: for example, enzyme kinetics studies explore the chemical mechanism of an enzyme's catalytic activity and its relative affinity for various possible substrate molecules. By contrast, in vivo experiments can provide information about the physiological role of a protein in the context of a cell or even a whole organism . In silico studies use computational methods to study proteins. Proteins may be purified from other cellular components using

1853-411: A protein is defined by the sequence of a gene, which is encoded in the genetic code . In general, the genetic code specifies 20 standard amino acids; but in certain organisms the genetic code can include selenocysteine and—in certain archaea — pyrrolysine . Shortly after or even during synthesis, the residues in a protein are often chemically modified by post-translational modification , which alters

1962-542: A protein that fold into distinct structural units. Domains usually also have specific functions, such as enzymatic activities (e.g. kinase ) or they serve as binding modules (e.g. the SH3 domain binds to proline-rich sequences in other proteins). Short amino acid sequences within proteins often act as recognition sites for other proteins. For instance, SH3 domains typically bind to short PxxP motifs (i.e. 2 prolines [P], separated by two unspecified amino acids [x], although

2071-486: A role in biological recognition phenomena involving cells and proteins. Receptors and hormones are highly specific binding proteins. Transmembrane proteins can also serve as ligand transport proteins that alter the permeability of the cell membrane to small molecules and ions. The membrane alone has a hydrophobic core through which polar or charged molecules cannot diffuse . Membrane proteins contain internal channels that allow such molecules to enter and exit

2180-582: A role in removing oxidative DNA damages . Protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues . Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions , DNA replication , responding to stimuli , providing structure to cells and organisms , and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which

2289-406: A series of purification steps may be necessary to obtain protein sufficiently pure for laboratory applications. To simplify this process, genetic engineering is often used to add chemical features to proteins that make them easier to purify without affecting their structure or activity. Here, a "tag" consisting of a specific amino acid sequence, often a series of histidine residues (a " His-tag "),

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2398-441: A simple example of a complementation test, suppose a geneticist is interested in studying two strains of white-eyed flies of the species Drosophila melanogaster , more commonly known as the common fruit fly. In this species, wild-type flies have red eyes, and eye color is known to be related to two genes, A and B. Each of these genes has two alleles, a dominant one that codes for a working protein ( A and B respectively) and

2507-432: A solution known as a crude lysate . The resulting mixture can be purified using ultracentrifugation , which fractionates the various cellular components into fractions containing soluble proteins; membrane lipids and proteins; cellular organelles , and nucleic acids . Precipitation by a method known as salting out can concentrate the proteins from this lysate. Various types of chromatography are then used to isolate

2616-441: A variety of techniques such as ultracentrifugation , precipitation , electrophoresis , and chromatography ; the advent of genetic engineering has made possible a number of methods to facilitate purification. To perform in vitro analysis, a protein must be purified away from other cellular components. This process usually begins with cell lysis , in which a cell's membrane is disrupted and its internal contents released into

2725-415: A wild-type phenotype. When complementation between two mutants defective in the same gene is measured, it is generally found that there is either no complementation or the complementation phenotype is intermediate between the mutant and wild-type phenotypes. Intragenic complementation (also called inter-allelic complementation) has been demonstrated in many different genes in a variety of organisms including

2834-663: 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 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 breaks in DNA, and in

2943-432: Is a rare recessive disorder in which affected individuals undergo rapid neurologic decline and indications of accelerated aging. A very severe case of such disabling mutations is F231L mutation in the tandem helix-hairpin-helix domain of ERCC1 at its interface with XPF. It is shown that this single mutation is very important for the stability of the ERCC1-XPF complex. This Phenylalanine residue is assisting ERCC1 to accommodate

3052-457: Is achieved by binding to AT-rich regions in the DNA and/or direct interaction with several transcription factors. HMGA2 targets and modifies the chromatin architecture at the ERCC1 gene, reducing its expression. Hypermethylation of the promoter for let-7a miRNA reduces its expression and this allows hyperexpression of HMGA2. Hyperexpression of HMGA2 can then reduce expression of ERCC1. Thus, there are three mechanisms that may be responsible for

3161-413: Is attached to one terminus of the protein. As a result, when the lysate is passed over a chromatography column containing nickel , the histidine residues ligate the nickel and attach to the column while the untagged components of the lysate pass unimpeded. A number of different tags have been developed to help researchers purify specific proteins from complex mixtures. Genetic complementation For

3270-479: Is dependent on the function of ERCC1-XPF in the host cell. Mammalian cells carrying mutations in ERCC1 or XPF are especially sensitive to agents that cause DNA interstrand crosslinks. 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

3379-562: Is dictated by the nucleotide sequence of their genes , and which usually results in protein folding into a specific 3D structure that determines its activity. A linear chain of amino acid residues is called a polypeptide . A protein contains at least one long polypeptide. Short polypeptides, containing less than 20–30 residues, are rarely considered to be proteins and are commonly called peptides . The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in

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3488-628: Is found in hard or filamentous structures such as hair , nails , feathers , hooves , and some animal shells . Some globular proteins can also play structural functions, for example, actin and tubulin are globular and soluble as monomers, but polymerize to form long, stiff fibers that make up the cytoskeleton , which allows the cell to maintain its shape and size. Other proteins that serve structural functions are motor proteins such as myosin , kinesin , and dynein , which are capable of generating mechanical forces. These proteins are crucial for cellular motility of single celled organisms and

3597-469: Is higher in prokaryotes than eukaryotes and can reach up to 20 amino acids per second. The process of synthesizing a protein from an mRNA template is known as translation . The mRNA is loaded onto the ribosome and is read three nucleotides at a time by matching each codon to its base pairing anticodon located on a transfer RNA molecule, which carries the amino acid corresponding to the codon it recognizes. The enzyme aminoacyl tRNA synthetase "charges"

3706-437: Is incompatible with viability of mice, and no human individuals have been found with complete (homozygous) deletion of ERCC1. Rare individuals in the human population harbor inherited mutations that impair the function of ERCC1. When the normal genes are absent, these mutations can lead to human syndromes, including Cockayne syndrome (CS) and COFS . ERCC1 and ERCC4 are the gene names assigned in mammalian genomes, including

3815-461: Is inefficient for polypeptides longer than about 300 amino acids, and the synthesized proteins may not readily assume their native tertiary structure . Most chemical synthesis methods proceed from C-terminus to N-terminus, opposite the biological reaction. Most proteins fold into unique 3D structures. The shape into which a protein naturally folds is known as its native conformation . Although many proteins can fold unassisted, simply through

3924-407: Is known as "non-allelic non-complementation" or "unlinked non-complementation"). This is an uncommon occurrence that depends on the type of mutants being investigated. Two mutations, for example, could be synthetically dominant negative . Transvection is another instance, in which a heterozygous combination of two alleles with mutations in distinct sections of the gene complement one other to restore

4033-404: Is often enormous—as much as 10 -fold increase in rate over the uncatalysed reaction in the case of orotate decarboxylase (78 million years without the enzyme, 18 milliseconds with the enzyme). The molecules bound and acted upon by enzymes are called substrates . Although enzymes can consist of hundreds of amino acids, it is usually only a small fraction of the residues that come in contact with

4142-484: Is one of the most frequent reductions of a DNA repair gene observed in a cancer. Deficiency in ERCC1 protein expression appears to be an early event in colon carcinogenesis , since ERCC1 was found to be deficient in 40% of the crypts within 10 cm on each side of colonic adenocarcinomas (within the early field defects from which the cancers likely arose). Cadmium (Cd) and its compounds are well-known human carcinogens . During Cd-induced malignant transformation,

4251-442: Is proposed to be adaptive because it facilitates recombinational repair of DNA damages that are otherwise difficult to repair. Outcrossing is proposed to be adaptive because it facilitates complementation, that is the masking of deleterious recessive alleles (also see heterosis ). The benefit of masking deleterious alleles has been proposed to be a major factor in the maintenance of sexual reproduction among eukaryotes. Further,

4360-763: Is reduced or absent in 84% to 100% of colorectal cancers , and lower expression of ERCC1 has been reported as being associated with unfavorable prognosis in patients undergoing treatment with oxaliplatin. The promoter of ERCC1 is methylated in 38% of gliomas, resulting in reduced mRNA and protein expression . The promoter of ERCC1 was located in the DNA 5 kilobases upstream of the protein coding region. Frequencies of epigenetic reductions of nine other DNA repair genes have been evaluated in various cancers and range from 2% ( OGG1 in papillary thyroid cancer) to 88% and 90% ( MGMT in gastric and colon cancers, respectively). Thus, reduction of protein expression of ERCC1 in 84% to 100% of colon cancers indicates that reduced ERCC1

4469-474: 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 tail is also needed in a mechanistically distinct subpathway of non-homologous end-joining, dependent on the Ku proteins. Homologous integration of DNA, an important technique for genetic manipulation,

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4578-486: Is the code for methionine . Because DNA contains four nucleotides, the total number of possible codons is 64; hence, there is some redundancy in the genetic code, with some amino acids specified by more than one codon. Genes encoded in DNA are first transcribed into pre- messenger RNA (mRNA) by proteins such as RNA polymerase . Most organisms then process the pre-mRNA (also known as a primary transcript ) using various forms of post-transcriptional modification to form

4687-502: Is the tendency for hybrid individuals to exceed their purebred parents in size and vigor. The phenomenon has long been known in animals and plants. Heterosis appears to be largely due to genetic complementation, that is the masking of deleterious recessive alleles in hybrid individuals. In general, the two fundamental aspects of sexual reproduction in eukaryotes are meiosis and outcrossing . These two aspects have been proposed to have two natural selective advantages, respectively. Meiosis

4796-556: Is thus a negative predictive marker, referring to how it will respond to a specific type of treatment. In colorectal cancer , clinical trials have not demonstrated the predictive ability of ERCC1 in oxaliplatin‐based treatment. Thus, European Society for Medical Oncology (ESMO) has not recommended ERCC1 testing prior to the use of oxaliplatin in routine practice. ERCC1 genotyping in humans has shown significant polymorphism at codon 118. These polymorphisms may have differential effects on platinum and mitomycin damage. ERCC1 protein expression

4905-481: The HMGA2 protein. HMGA proteins are characterized by three DNA-binding domains, called AT-hooks , and an acidic carboxy-terminal tail. HMGA proteins are chromatin architectural transcription factors that both positively and negatively regulate the transcription of a variety of genes. They do not display direct transcriptional activation capacity, but regulate gene expression by changing local DNA conformation. Regulation

5014-479: The MUS81 - EME1 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. This structure-specificity

5123-492: The amino acid leucine for which he found a (nearly correct) molecular weight of 131 Da . Early nutritional scientists such as the German Carl von Voit believed that protein was the most important nutrient for maintaining the structure of the body, because it was generally believed that "flesh makes flesh." Around 1862, Karl Heinrich Ritthausen isolated the amino acid glutamic acid . Thomas Burr Osborne compiled

5232-644: The muscle sarcomere , with a molecular mass of almost 3,000 kDa and a total length of almost 27,000 amino acids. Short proteins can also be synthesized chemically by a family of methods known as peptide synthesis , which rely on organic synthesis techniques such as chemical ligation to produce peptides in high yield. Chemical synthesis allows for the introduction of non-natural amino acids into polypeptide chains, such as attachment of fluorescent probes to amino acid side chains. These methods are useful in laboratory biochemistry and cell biology , though generally not for commercial applications. Chemical synthesis

5341-645: The sperm of many multicellular organisms which reproduce sexually . They also generate the forces exerted by contracting muscles and play essential roles in intracellular transport. A key question in molecular biology is how proteins evolve, i.e. how can mutations (or rather changes in amino acid sequence) lead to new structures and functions? Most amino acids in a protein can be changed without disrupting activity or function, as can be seen from numerous homologous proteins across species (as collected in specialized databases for protein families , e.g. PFAM ). In order to prevent dramatic consequences of mutations,

5450-497: The 1700s by Antoine Fourcroy and others, who often collectively called them " albumins ", or "albuminous materials" ( Eiweisskörper , in German). Gluten , for example, was first separated from wheat in published research around 1747, and later determined to exist in many plants. In 1789, Antoine Fourcroy recognized three distinct varieties of animal proteins: albumin , fibrin , and gelatin . Vegetable (plant) proteins studied in

5559-572: The 1950s, the Armour Hot Dog Company purified 1 kg of pure bovine pancreatic ribonuclease A and made it freely available to scientists; this gesture helped ribonuclease A become a major target for biochemical study for the following decades. The understanding of proteins as polypeptides , or chains of amino acids, came through the work of Franz Hofmeister and Hermann Emil Fischer in 1902. The central role of proteins as enzymes in living organisms that catalyzed reactions

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5668-498: The 20,000 or so proteins encoded by the human genome, only 6,000 are detected in lymphoblastoid cells. Proteins are assembled from amino acids using information encoded in genes. Each protein has its own unique amino acid sequence that is specified by the nucleotide sequence of the gene encoding this protein. The genetic code is a set of three-nucleotide sets called codons and each three-nucleotide combination designates an amino acid, for example AUG ( adenine – uracil – guanine )

5777-679: The DNA helix for a short distance on either side of the site of a 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. 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

5886-519: The EC number system provides a functional classification scheme. Similarly, the gene ontology classifies both genes and proteins by their biological and biochemical function, but also by their intracellular location. Sequence similarity is used to classify proteins both in terms of evolutionary and functional similarity. This may use either whole proteins or protein domains , especially in multi-domain proteins . Protein domains allow protein classification by

5995-593: The HIV virus, down-regulates ERCC1 protein expression. TAR miRNA allows ERCC1 mRNA to be transcribed, but acts at the p-body level to prevent translation of ERCC1 protein. (A p-body is a cytoplasmic granule “processing body” that interacts with miRNAs to repress translation or trigger degradation of target RNAs.) In breast cancer cell lines, almost one third (55/167) of miRNA promoters were targets for aberrant methylation ( epigenetic repression). In breast cancers themselves, methylation of let-7a-3/let-7b miRNA in particular

6104-709: The ability of many enzymes to bind and process multiple substrates . When mutations occur, the specificity of an enzyme can increase (or decrease) and thus its enzymatic activity. Thus, bacteria (or other organisms) can adapt to different food sources, including unnatural substrates such as plastic. Methods commonly used to study protein structure and function include immunohistochemistry , site-directed mutagenesis , X-ray crystallography , nuclear magnetic resonance and mass spectrometry . The activities and structures of proteins may be examined in vitro , in vivo , and in silico . In vitro studies of purified proteins in controlled environments are useful for learning how

6213-593: The active site residues for nuclease activity. 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,

6322-405: The addition of a single methyl group to a binding partner can sometimes suffice to nearly eliminate binding; for example, the aminoacyl tRNA synthetase specific to the amino acid valine discriminates against the very similar side chain of the amino acid isoleucine . Proteins can bind to other proteins as well as to small-molecule substrates. When proteins bind specifically to other copies of

6431-607: The alpha carbons are roughly coplanar . The other two dihedral angles in the peptide bond determine the local shape assumed by the protein backbone. The end with a free amino group is known as the N-terminus or amino terminus, whereas the end of the protein with a free carboxyl group is known as the C-terminus or carboxy terminus (the sequence of the protein is written from N-terminus to C-terminus, from left to right). The words protein , polypeptide, and peptide are

6540-531: The amino acid side chains in a protein that ultimately determines its three-dimensional structure and its chemical reactivity. The amino acids in a polypeptide chain are linked by peptide bonds . Once linked in the protein chain, an individual amino acid is called a residue, and the linked series of carbon, nitrogen, and oxygen atoms are known as the main chain or protein backbone. The peptide bond has two resonance forms that contribute some double-bond character and inhibit rotation around its axis, so that

6649-574: The binding of a substrate molecule to an enzyme's active site , or the physical region of the protein that participates in chemical catalysis. In solution, proteins also undergo variation in structure through thermal vibration and the collision with other molecules. Proteins can be informally divided into three main classes, which correlate with typical tertiary structures: globular proteins , fibrous proteins , and membrane proteins . Almost all globular proteins are soluble and many are enzymes. Fibrous proteins are often structural, such as collagen ,

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6758-570: The body of a multicellular organism. These proteins must have a high binding affinity when their ligand is present in high concentrations, but must also release the ligand when it is present at low concentrations in the target tissues. The canonical example of a ligand-binding protein is haemoglobin , which transports oxygen from the lungs to other organs and tissues in all vertebrates and has close homologs in every biological kingdom . Lectins are sugar-binding proteins which are highly specific for their sugar moieties. Lectins typically play

6867-558: The cell is as enzymes , which catalyse chemical reactions. Enzymes are usually highly specific and accelerate only one or a few chemical reactions. Enzymes carry out most of the reactions involved in metabolism , as well as manipulating DNA in processes such as DNA replication , DNA repair , and transcription . Some enzymes act on other proteins to add or remove chemical groups in a process known as posttranslational modification. About 4,000 reactions are known to be catalysed by enzymes. The rate acceleration conferred by enzymatic catalysis

6976-436: The cell surface and an effector domain within the cell, which may have enzymatic activity or may undergo a conformational change detected by other proteins within the cell. Antibodies are protein components of an adaptive immune system whose main function is to bind antigens , or foreign substances in the body, and target them for destruction. Antibodies can be secreted into the extracellular environment or anchored in

7085-752: The cell's machinery through the process of protein turnover . A protein's lifespan is measured in terms of its half-life and covers a wide range. They can exist for minutes or years with an average lifespan of 1–2 days in mammalian cells. Abnormal or misfolded proteins are degraded more rapidly either due to being targeted for destruction or due to being unstable. Like other biological macromolecules such as polysaccharides and nucleic acids , proteins are essential parts of organisms and participate in virtually every process within cells . Many proteins are enzymes that catalyse biochemical reactions and are vital to metabolism . Proteins also have structural or mechanical functions, such as actin and myosin in muscle and

7194-450: The cell. Many ion channel proteins are specialized to select for only a particular ion; for example, potassium and sodium channels often discriminate for only one of the two ions. Structural proteins confer stiffness and rigidity to otherwise-fluid biological components. Most structural proteins are fibrous proteins ; for example, collagen and elastin are critical components of connective tissue such as cartilage , and keratin

7303-621: The chemical properties of their amino acids, others require the aid of molecular chaperones to fold into their native states. Biochemists often refer to four distinct aspects of a protein's structure: Proteins are not entirely rigid molecules. In addition to these levels of structure, proteins may shift between several related structures while they perform their functions. In the context of these functional rearrangements, these tertiary or quaternary structures are usually referred to as " conformations ", and transitions between them are called conformational changes. Such changes are often induced by

7412-441: The chief actors within the cell, said to be carrying out the duties specified by the information encoded in genes. With the exception of certain types of RNA , most other biological molecules are relatively inert elements upon which proteins act. Proteins make up half the dry weight of an Escherichia coli cell, whereas other macromolecules such as DNA and RNA make up only 3% and 20%, respectively. The set of proteins expressed in

7521-452: 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. During nucleotide excision repair, several protein complexes cooperate to recognize damaged DNA and locally separate

7630-490: The construction of enormously complex signaling networks. As interactions between proteins are reversible, and depend heavily on the availability of different groups of partner proteins to form aggregates that are capable to carry out discrete sets of function, study of the interactions between specific proteins is a key to understand important aspects of cellular function, and ultimately the properties that distinguish particular cell types. The best-known role of proteins in

7739-503: The crosslink and initiate repair. Alternatively, a double-strand break may be made in the DNA near the ICL, and subsequent homologous recombination repair may involve ERCC1-XPF action. Although not the only nuclease involved, ERCC1–XPF is required for ICL repair during several phases of the cell cycle. A few patients with severely disabling ERCC1 mutations that cause cerebro-oculo-facio-skeletal syndrome (COFS) have been reported. COFS syndrome

7848-408: The derivative unit kilodalton (kDa). The average size of a protein increases from Archaea to Bacteria to Eukaryote (283, 311, 438 residues and 31, 34, 49 kDa respectively) due to a bigger number of protein domains constituting proteins in higher organisms. For instance, yeast proteins are on average 466 amino acids long and 53 kDa in mass. The largest known proteins are the titins , a component of

7957-402: The endonuclease active site and is involved in DNA binding and additional protein–protein interactions. The ERCC4/XPF protein consists of two conserved domains separated by a less conserved region in the middle. The N-terminal region 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

8066-451: The erroneous conclusion that they might be composed of a single type of (very large) molecule. The term "protein" to describe these molecules was proposed by Mulder's associate Berzelius; protein is derived from the Greek word πρώτειος ( proteios ), meaning "primary", "in the lead", or "standing in front", + -in . Mulder went on to identify the products of protein degradation such as

8175-468: The fungi Neurospora crassa , Saccharomyces cerevisiae , and Schizosaccharomyces pombe ; the bacterium Salmonella typhimurium ; and the virus bacteriophage T4 . In several such studies, numerous mutations defective in the same gene were isolated and mapped in a linear order based on recombination frequencies to form a genetic map of the gene. Separately, the mutants were tested in pairwise combinations to measure complementation. An analysis of

8284-438: The fungus Neurospora crassa led to the development of the one-gene-one-enzyme concept that provided the foundation for the subsequent development of molecular genetics. The complementation test was one of the main tools used in the early Neurospora work, because it was easy to do, and allowed the investigator to determine whether any two nutritional mutants were defective in the same or different genes. The complementation test

8393-459: The gene was called “Excision repair cross-complementing 1”. Multiple independent complementation groups of Chinese hamster ovary (CHO) cells were isolated, and this gene restored UV resistance to cells of complementation group 1. The human ERCC1 gene encodes the ERCC1 protein of 297 amino acids with a molecular mass of about 32,500 daltons. Genes similar to ERCC1 with equivalent functions (orthologs) are found in other eukaryotic genomes. Some of

8502-444: The human genome ( Homo sapiens ). Similar genes with similar functions are found in all eukaryotic organisms. The genomic DNA for ERCC1 was the first human DNA repair gene to be isolated by molecular cloning. The original method was by transfer of fragments of the human genome to ultraviolet light (UV)-sensitive mutant cell lines derived from Chinese hamster ovary cells . Reflecting this cross-species genetic complementation method,

8611-534: The late 1700s and early 1800s included gluten , plant albumin , gliadin , and legumin . Proteins were first described by the Dutch chemist Gerardus Johannes Mulder and named by the Swedish chemist Jöns Jacob Berzelius in 1838. Mulder carried out elemental analysis of common proteins and found that nearly all proteins had the same empirical formula , C 400 H 620 N 100 O 120 P 1 S 1 . He came to

8720-402: The level of overall mRNA transcription, splice variation nor transcription start point of mRNA correlates with protein level of ERCC1. The rate of ERCC1 protein turnover also does not correlate with ERCC1 protein level. A translational level control of ERCC1, due to a microRNA (miRNA), has been shown during HIV viral infection. A trans-activation response element (TAR) miRNA, coded for by

8829-657: The low level of protein expression of ERCC1 in 84% to 100% of sporadic colon cancers. From results in gliomas and in cadmium carcinogenesis, methylation of the ERCC1 promoter may be a factor. One or more miRNAs that repress ERCC1 may be a factor. And epigenetically reduced let-7a miRNA allowing hyperexpression of HMGA2 could also reduce protein expression of ERCC1 in colon cancers. Which epigenetic mechanism occurs most frequently, or whether multiple epigenetic mechanisms reduce ERCC1 protein expression in colon cancers has not been determined. DNA repair -deficient Ercc1 mutant mice show numerous features of accelerated aging, and have

8938-478: The major component of connective tissue, or keratin , the protein component of hair and nails. Membrane proteins often serve as receptors or provide channels for polar or charged molecules to pass through the cell membrane . A special case of intramolecular hydrogen bonds within proteins, poorly shielded from water attack and hence promoting their own dehydration , are called dehydrons . Many proteins are composed of several protein domains , i.e. segments of

9047-443: The mature mRNA, which is then used as a template for protein synthesis by the ribosome . In prokaryotes the mRNA may either be used as soon as it is produced, or be bound by a ribosome after having moved away from the nucleoid . In contrast, eukaryotes make mRNA in the cell nucleus and then translocate it across the nuclear membrane into the cytoplasm , where protein synthesis then takes place. The rate of protein synthesis

9156-405: The membranes of specialized B cells known as plasma cells . Whereas enzymes are limited in their binding affinity for their substrates by the necessity of conducting their reaction, antibodies have no such constraints. An antibody's binding affinity to its target is extraordinarily high. Many ligand transport proteins bind particular small biomolecules and transport them to other locations in

9265-453: The most studied gene orthologs include RAD10 in the budding yeast Saccharomyces cerevisiae , and swi10+ in the fission yeast Schizosaccharomyces pombe . 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

9374-971: The nervous system, liver and kidney (see DNA damage theory of aging ). When Ercc1 mutant mice were subjected to dietary restriction their response closely resembled the beneficial response to dietary restriction of wild-type mice. Dietary restriction extended the lifespan of the Ercc1 mutant mice from 10 to 35 weeks for males and from 13 to 39 weeks for females. It appears that in Ercc1 mutant mice dietary restriction while delaying aging also attenuates accumulation of genome-wide DNA damage and preserves transcriptional output, likely contributing to improved cell viability. Both male and female Ercc1 -deficient mice are infertile . The DNA repair function of Ercc1 appears to be required in both male and female germ cells at all stages of their maturation. The testes of Ercc1 -deficient mice have an increased level of 8-oxoguanine in their DNA , suggesting that Ercc1 may have

9483-496: The nobel prize in 1972, solidified the thermodynamic hypothesis of protein folding, according to which the folded form of a protein represents its free energy minimum. With the development of X-ray crystallography , it became possible to determine protein structures as well as their sequences. The first protein structures to be solved were hemoglobin by Max Perutz and myoglobin by John Kendrew , in 1958. The use of computers and increasing computing power also supported

9592-500: The order of 50,000 to 1 million. By contrast, eukaryotic cells are larger and thus contain much more protein. For instance, yeast cells have been estimated to contain about 50 million proteins and human cells on the order of 1 to 3 billion. The concentration of individual protein copies ranges from a few molecules per cell up to 20 million. Not all genes coding proteins are expressed in most cells and their number depends on, for example, cell type and external stimuli. For instance, of

9701-488: The other 'AA bb'), resulting in a heterozygous genotype ('AaBb') in the progeny that produces a different phenotype from the parents. Non-complementation is observed when both strains share the same homozygous mutation, such as 'aaBB', 'AAbb', or 'aabb', leading to progeny with a phenotype identical to the parent strains. Complementation tests can also be carried out with haploid eukaryotes such as fungi , with bacteria, and with viruses such as bacteriophage . Research on

9810-440: The physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. Some proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors . Proteins can also work together to achieve a particular function, and they often associate to form stable protein complexes . Once formed, proteins only exist for a certain period and are then degraded and recycled by

9919-424: The process of cell signaling and signal transduction . Some proteins, such as insulin , are extracellular proteins that transmit a signal from the cell in which they were synthesized to other cells in distant tissues . Others are membrane proteins that act as receptors whose main function is to bind a signaling molecule and induce a biochemical response in the cell. Many receptors have a binding site exposed on

10028-432: The promoter regions of ERCC1 , as well as of h MSH2 , XRCC1 , and h OGG1 , were heavily methylated and both the messenger RNA and proteins of these DNA repair genes were progressively reduced. DNA damage also increased with Cd-induced transformation. Reduction of protein expression of ERCC1 in progression to sporadic cancer is unlikely to be due to mutation. While germ line (familial) mutations in DNA repair genes cause

10137-534: The protein or proteins of interest based on properties such as molecular weight, net charge and binding affinity. The level of purification can be monitored using various types of gel electrophoresis if the desired protein's molecular weight and isoelectric point are known, by spectroscopy if the protein has distinguishable spectroscopic features, or by enzyme assays if the protein has enzymatic activity. Additionally, proteins can be isolated according to their charge using electrofocusing . For natural proteins,

10246-427: The proteins in the cytoskeleton , which form a system of scaffolding that maintains cell shape. Other proteins are important in cell signaling, immune responses , cell adhesion , and the cell cycle . In animals, proteins are needed in the diet to provide the essential amino acids that cannot be synthesized . Digestion breaks the proteins down for metabolic use. Proteins have been studied and recognized since

10355-495: The repair of “crosslink” damage that harmfully links the two DNA strands. Cells with disabling mutations in ERCC1 are more sensitive than normal to particular DNA damaging agents, including ultraviolet (UV) radiation and to chemicals that cause crosslinking between DNA strands. Genetically engineered mice with disabling mutations in ERCC1 have defects in DNA repair, accompanied by metabolic stress-induced changes in physiology that result in premature aging. Complete deletion of ERCC1

10464-444: The results from such studies led to the conclusion that intragenic complementation, in general, arises from the interaction of differently defective polypeptide monomers to form an aggregate called a “multimer.” Genes that encode multimer-forming polypeptides appear to be common. One interpretation of the data is that polypeptide monomers are often aligned in the multimer in such a way that mutant polypeptides defective at nearby sites in

10573-582: The same molecule, they can oligomerize to form fibrils; this process occurs often in structural proteins that consist of globular monomers that self-associate to form rigid fibers. Protein–protein interactions also regulate enzymatic activity, control progression through the cell cycle , and allow the assembly of large protein complexes that carry out many closely related reactions with a common biological function. Proteins can also bind to, or even be integrated into, cell membranes. The ability of binding partners to induce conformational changes in proteins allows

10682-581: The sample, allowing scientists to obtain more information and analyze larger structures. Computational protein structure prediction of small protein structural domains has also helped researchers to approach atomic-level resolution of protein structures. As of April 2024 , the Protein Data Bank contains 181,018 X-ray, 19,809 EM and 12,697 NMR protein structures. Proteins are primarily classified by sequence and structure, although other classifications are commonly used. Especially for enzymes

10791-517: The selective advantage of complementation that arises from outcrossing may largely account for the general avoidance of inbreeding in nature (e.g. see articles kin recognition , inbreeding depression , and incest taboo ). Used by quantitative genetics to uncover recessive mutants. Here one takes deficiencies and crosses them to a haplotype that is believed to contain the recessive mutant. These rules (patterns) are not without exceptions. Non-allelic mutants may occasionally fail to complement (this

10900-430: The sequencing of complex proteins. In 1999, Roger Kornberg succeeded in sequencing the highly complex structure of RNA polymerase using high intensity X-rays from synchrotrons . Since then, cryo-electron microscopy (cryo-EM) of large macromolecular assemblies has been developed. Cryo-EM uses protein samples that are frozen rather than crystals, and beams of electrons rather than X-rays. It causes less damage to

11009-405: The substrate, and an even smaller fraction—three to four residues on average—that are directly involved in catalysis. The region of the enzyme that binds the substrate and contains the catalytic residues is known as the active site . Dirigent proteins are members of a class of proteins that dictate the stereochemistry of a compound synthesized by other enzymes. Many proteins are involved in

11118-716: The surrounding amino acids may determine the exact binding specificity). Many such motifs has been collected in the Eukaryotic Linear Motif (ELM) database. Topology of a protein describes the entanglement of the backbone and the arrangement of contacts within the folded chain. Two theoretical frameworks of knot theory and Circuit topology have been applied to characterise protein topology. Being able to describe protein topology opens up new pathways for protein engineering and pharmaceutical development, and adds to our understanding of protein misfolding diseases such as neuromuscular disorders and cancer. Proteins are

11227-400: The tRNA molecules with the correct amino acids. The growing polypeptide is often termed the nascent chain . Proteins are always biosynthesized from N-terminus to C-terminus . The size of a synthesized protein can be measured by the number of amino acids it contains and by its total molecular mass , which is normally reported in units of daltons (synonymous with atomic mass units ), or

11336-472: The tertiary structure of the protein, which defines the binding site pocket, and by the chemical properties of the surrounding amino acids' side chains. Protein binding can be extraordinarily tight and specific; for example, the ribonuclease inhibitor protein binds to human angiogenin with a sub-femtomolar dissociation constant (<10 M) but does not bind at all to its amphibian homolog onconase (> 1 M). Extremely minor chemical changes such as

11445-472: Was insulin , by Frederick Sanger , in 1949. Sanger correctly determined the amino acid sequence of insulin, thus conclusively demonstrating that proteins consisted of linear polymers of amino acids rather than branched chains, colloids , or cyclols . He won the Nobel Prize for this achievement in 1958. Christian Anfinsen 's studies of the oxidative folding process of ribonuclease A, for which he won

11554-464: Was also used in the early development of molecular genetics when bacteriophage T4 was one of the main objects of study. In this case the test depends on mixed infections of host bacterial cells with two different bacteriophage mutant types. Its use was key to defining most of the genes of the virus, and provided the foundation for the study of such fundamental processes as DNA replication and repair, and how molecular machines are constructed. Heterosis

11663-476: Was found. This indicates that let-7a-3/let-7b can be epigenetically repressed. Repression of let-7a can cause repression of ERCC1 expression through an intermediary step involving the HMGA2 gene. The let-7a miRNA normally represses the HMGA2 gene, and in normal adult tissues, almost no HMGA2 protein is present. (See also Let-7 microRNA precursor .) Reduction or absence of let-7a miRNA allows high expression of

11772-472: 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

11881-581: Was not fully appreciated until 1926, when James B. Sumner showed that the enzyme urease was in fact a protein. Linus Pauling is credited with the successful prediction of regular protein secondary structures based on hydrogen bonding , an idea first put forth by William Astbury in 1933. Later work by Walter Kauzmann on denaturation , based partly on previous studies by Kaj Linderstrøm-Lang , contributed an understanding of protein folding and structure mediated by hydrophobic interactions . The first protein to have its amino acid chain sequenced

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