Nucleotide excision repair
Encyclopedia
Nucleotide excision repair is a DNA repair
mechanism. DNA
constantly requires repair due to damage that can occur to bases from a vast variety of sources including chemicals, radiation and other mutagens. Nucleotide excision repair (NER) is a particularly important mechanism by which the cell can prevent unwanted mutations by removing the vast majority of UV-induced DNA damage (mostly in the form of thymine dimers and 6-4-photoproducts). The importance of this repair mechanism is evidenced by the severe human diseases that result from in-born genetic mutations of NER proteins including Xeroderma pigmentosum
and Cockayne's syndrome
. While the base excision repair
machinery can recognize specific lesions in the DNA it can correct only damaged bases that can be removed by a specific glycosylase, the nucleotide excision repair enzymes recognize bulky distortions in the shape of the DNA double helix. Recognition of these distortions leads to the removal of a short single-stranded DNA segment that includes the lesion, creating a single-strand gap in the DNA, which is subsequently filled in by DNA polymerase, which uses the undamaged strand as a template. NER can be divided into two subpathways (Global genomic NER and Transcription coupled NER) that differ only in their recognition of helix-distorting DNA damage.
by the UvrABC endonuclease
enzyme complex, which consists of four Uvr proteins: UvrA, UvrB, UvrC, and DNA helicase II (sometimes also known as UvrD in this complex). First, a UvrA-UvrB complex scans the DNA, with the UvrA subunit recognizing distortions in the helix, caused for example by pyrimidine dimers. When the complex recognizes such a distortion, the UvrA subunit leaves and an UvrC protein comes in and binds to the UvrB monomer and, hence, forms a new UvrBC dimer. UvrB cleaves a phosphodiester bond
4 nucleotides downstream of the DNA damage, and the UvrC cleaves a phosphodiester bond 8 nucleotides upstream of the DNA damage and created 12 nucleotide excised segment. DNA helicase II (sometimes called UvrD) then comes in and removes the excised segment by actively breaking the hydrogen bonds between the complementary bases. The resultant gap is then filled in using DNA polymerase
I and DNA ligase
. The basic excision process is very similar in higher cells, but these cells usually involve many more proteins – E.coli is a simple example.
, XPB
, XPC
, XPD, XPE, XPF, and XPG all derive from Xeroderma pigmentosum
and CSA and CSB
represent proteins linked to Cockayne syndrome
. Additionally, the proteins ERCC1
, RPA
, RAD23A
, RAD23B
, and others also participate in nucleotide excision repair.
As described below, nucleotide excision repair can be categorized into two classes, global genome NER (GG-NER) and Transcription Coupled NER (TC-NER). Two different sets of proteins are involved in the distortion and recognition of the DNA damage in the two types of NER. In GG-NER, the XPC
-Rad23B complex is responsible for distortion recognition, and DDB1 and DDB2 (XPE) can also recognize some types of damage caused by UV light. Additionally, XPA performs a function in damage recognition that is as yet poorly defined. In TC-NER, CS proteins CSA and CSB bind some types of DNA damage instead of XPC-Rad23B.
The subsequent steps in GG-NER and TC-NER are similar to each other and to those in NER in prokaryotes. XPB and XPD, which are subunits of transcription factor TFIIH have helicase
activity and unwind the DNA at the sites of damages. XPG protein has a structure-specific endonuclease
activity, which makes an incision 3’ to the damaged DNA. Subsequently XPF protein, which is associated with ERCC1 makes the 5' incision during the NER. The dual-incision leads to the removal of a ssDNA with a single strand gap of 25~30 nucleotides.
The resulting gap in DNA is filled by DNA polymerase
δ or ε by copying the undamaged strand. Proliferating Cell Nuclear Antigen (PCNA) assists the DNA polymerase in the reaction, and Replication protein A
(RPA) protects the other DNA strand from degradation during NER. Finally, DNA ligase
seals the nicks to finish NER.
A 2002 paper published by Frit et al. showed that Global Genome Repair can be enhanced near transcribed regions. Their main conclusion is that the binding of transcription factors led to an increase in nucleotide excision repair on nearby regions of DNA. They showed that this enhancement was distinct from Transcription Coupled Repair in two ways: Repair was occurring on both the template and non-template strands. Secondly, when Pol-II activity was blocked, either by α-amanitin or modification of the TATA box
, the repair still occurred. Through digestions with micrococcal nuclease
they determined that the binding of transcription factors led to local chromatin
remodeling, likely giving NER proteins greater access to the DNA.
At any given time, most of the genome in an organism is not undergoing transcription. The genome-wide process which repairs damage in both transcribed and untranscribed DNA strands in active and inactive genes is called GGR – this process is not dependent on transcription.
TC-NER and GGR are two subpathways of NER, differing only in the initial steps of DNA damage recognition. The principal difference is that TC-NER does not require XPC or DDB proteins for distortion recognition in mammalian cells. TC-NER is instead thought to be initiated when RNA polymerase stalls at a lesion in DNA. In TC-NER the blocked RNA polymerase serves as a damage recognition signal, replacing the need for the distortion recognition properties of the XPC-RAD23B and DDB complexes. Subsequent steps in TC-NER utilize the NER factors XPA, TFIIH, and RPA as well as the nucleases ECC1-XPF and XPG for dual incision at a lesion. The repair patch size for mammalian cells in vivo is around 30 nucleotides, indistinguishable from the repair patch size for GGR.
Excision Enzyme cuts at the Nucleotide excision repair. (L Bridgewater, experiment TAB)
DNA repair
DNA repair refers to 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 UV light and radiation can cause DNA damage, resulting in as many as 1...
mechanism. DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
constantly requires repair due to damage that can occur to bases from a vast variety of sources including chemicals, radiation and other mutagens. Nucleotide excision repair (NER) is a particularly important mechanism by which the cell can prevent unwanted mutations by removing the vast majority of UV-induced DNA damage (mostly in the form of thymine dimers and 6-4-photoproducts). The importance of this repair mechanism is evidenced by the severe human diseases that result from in-born genetic mutations of NER proteins including Xeroderma pigmentosum
Xeroderma pigmentosum
Xeroderma pigmentosum, or XP, is an autosomal recessive genetic disorder of DNA repair in which the ability to repair damage caused by ultraviolet light is deficient. In extreme cases, all exposure to sunlight must be forbidden, no matter how small. Multiple basal cell carcinomas and other skin...
and Cockayne's syndrome
Cockayne syndrome
Cockayne syndrome is a rare autosomal recessive, congenital disorder characterized by growth failure, impaired development of the nervous system, abnormal sensitivity to sunlight , and premature aging...
. While the base excision repair
Base excision repair
In biochemistry and genetics, base excision repair is a cellular mechanism that repairs damaged DNA throughout the cell cycle. It is responsible primarily for removing small, non-helix-distorting base lesions from the genome. The related nucleotide excision repair pathway repairs bulky...
machinery can recognize specific lesions in the DNA it can correct only damaged bases that can be removed by a specific glycosylase, the nucleotide excision repair enzymes recognize bulky distortions in the shape of the DNA double helix. Recognition of these distortions leads to the removal of a short single-stranded DNA segment that includes the lesion, creating a single-strand gap in the DNA, which is subsequently filled in by DNA polymerase, which uses the undamaged strand as a template. NER can be divided into two subpathways (Global genomic NER and Transcription coupled NER) that differ only in their recognition of helix-distorting DNA damage.
Uvr Proteins
The process of nucleotide excision repair is controlled in E. coliEscherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
by the UvrABC endonuclease
UvrABC endonuclease
UvrABC endonuclease is a multienzyme complex in E.coli bacteria involved in DNA repair mechanism by nucleotide excision repair, and it is, therefore, sometimes called an excinuclease...
enzyme complex, which consists of four Uvr proteins: UvrA, UvrB, UvrC, and DNA helicase II (sometimes also known as UvrD in this complex). First, a UvrA-UvrB complex scans the DNA, with the UvrA subunit recognizing distortions in the helix, caused for example by pyrimidine dimers. When the complex recognizes such a distortion, the UvrA subunit leaves and an UvrC protein comes in and binds to the UvrB monomer and, hence, forms a new UvrBC dimer. UvrB cleaves a phosphodiester bond
Phosphodiester bond
A phosphodiester bond is a group of strong covalent bonds between a phosphate group and two 5-carbon ring carbohydrates over two ester bonds. Phosphodiester bonds are central to all known life, as they make up the backbone of each helical strand of DNA...
4 nucleotides downstream of the DNA damage, and the UvrC cleaves a phosphodiester bond 8 nucleotides upstream of the DNA damage and created 12 nucleotide excised segment. DNA helicase II (sometimes called UvrD) then comes in and removes the excised segment by actively breaking the hydrogen bonds between the complementary bases. The resultant gap is then filled in using DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
I and DNA ligase
DNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
. The basic excision process is very similar in higher cells, but these cells usually involve many more proteins – E.coli is a simple example.
Nucleotide Excision Repair in Eukaryotes
Nucleotide excision repair has more complexity in eukaryotes. But the general principles upon which it operates are similar. There are 9 major proteins involved in NER in mammalian cells and their names come from the diseases associated with the deficiencies in those proteins. XPAXPA
DNA repair protein complementing XP-A cells is a protein that in humans is encoded by the XPA gene.-Interactions:XPA has been shown to interact with ERCC1, Replication protein A1 and XAB2.-External Links:* -Further reading:...
, XPB
XPB
XPB is an ATP dependent human DNA helicase that is a part of the TFIIH transcription factor complex.-Structure:The 3D structure of the archeael homologue of XPB has been solved by X-ray crystallography by Dr...
, XPC
XPC (gene)
Xeroderma pigmentosum, complementation group C, also known as XPC, is a protein which in humans is encoded by the XPC gene. XPC is involved in the recognition of bulky DNA adducts in nucleotide excision repair.- Function :...
, XPD, XPE, XPF, and XPG all derive from Xeroderma pigmentosum
Xeroderma pigmentosum
Xeroderma pigmentosum, or XP, is an autosomal recessive genetic disorder of DNA repair in which the ability to repair damage caused by ultraviolet light is deficient. In extreme cases, all exposure to sunlight must be forbidden, no matter how small. Multiple basal cell carcinomas and other skin...
and CSA and CSB
ERCC6
DNA excision repair protein ERCC-6 is a protein that in humans is encoded by the ERCC6 gene.-External links:*...
represent proteins linked to Cockayne syndrome
Cockayne syndrome
Cockayne syndrome is a rare autosomal recessive, congenital disorder characterized by growth failure, impaired development of the nervous system, abnormal sensitivity to sunlight , and premature aging...
. Additionally, the proteins ERCC1
ERCC1
DNA excision repair protein ERCC-1 is a protein that in humans is encoded by the ERCC1 gene.- Function :The function of the ERCC1 protein is predominantly in nucleotide excision repair of damaged DNA...
, RPA
Replication protein A
Replication protein A is a protein that binds single-stranded DNA in eukaryotic cells. During DNA replication, RPA prevents single-stranded DNA from winding back on itself or from forming secondary structures. This keeps DNA unwound for the polymerase to replicate it...
, RAD23A
RAD23A
UV excision repair protein RAD23 homolog A is a protein that in humans is encoded by the RAD23A gene.-Interactions:RAD23A has been shown to interact with PSMD4, Sequestosome 1 and Ataxin 3.-Further reading:...
, RAD23B
RAD23B
UV excision repair protein RAD23 homolog B is a protein that in humans is encoded by the RAD23B gene.-Interactions:RAD23B has been shown to interact with PSMD4 and Ataxin 3.-Further reading:...
, and others also participate in nucleotide excision repair.
As described below, nucleotide excision repair can be categorized into two classes, global genome NER (GG-NER) and Transcription Coupled NER (TC-NER). Two different sets of proteins are involved in the distortion and recognition of the DNA damage in the two types of NER. In GG-NER, the XPC
XPC (gene)
Xeroderma pigmentosum, complementation group C, also known as XPC, is a protein which in humans is encoded by the XPC gene. XPC is involved in the recognition of bulky DNA adducts in nucleotide excision repair.- Function :...
-Rad23B complex is responsible for distortion recognition, and DDB1 and DDB2 (XPE) can also recognize some types of damage caused by UV light. Additionally, XPA performs a function in damage recognition that is as yet poorly defined. In TC-NER, CS proteins CSA and CSB bind some types of DNA damage instead of XPC-Rad23B.
The subsequent steps in GG-NER and TC-NER are similar to each other and to those in NER in prokaryotes. XPB and XPD, which are subunits of transcription factor TFIIH have helicase
Helicase
Helicases are a class of enzymes vital to all living organisms. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands using energy derived from ATP hydrolysis.-Function:Many cellular processes Helicases are a...
activity and unwind the DNA at the sites of damages. XPG protein has a structure-specific endonuclease
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
activity, which makes an incision 3’ to the damaged DNA. Subsequently XPF protein, which is associated with ERCC1 makes the 5' incision during the NER. The dual-incision leads to the removal of a ssDNA with a single strand gap of 25~30 nucleotides.
The resulting gap in DNA is filled by DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
δ or ε by copying the undamaged strand. Proliferating Cell Nuclear Antigen (PCNA) assists the DNA polymerase in the reaction, and Replication protein A
Replication protein A
Replication protein A is a protein that binds single-stranded DNA in eukaryotic cells. During DNA replication, RPA prevents single-stranded DNA from winding back on itself or from forming secondary structures. This keeps DNA unwound for the polymerase to replicate it...
(RPA) protects the other DNA strand from degradation during NER. Finally, DNA ligase
DNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
seals the nicks to finish NER.
Global genomic NER
Global genomic NER repairs damage in both transcribed and untranscribed DNA strands in active and inactive genes throughout the genome. This pathway employs several 'damage sensing' proteins including the DNA-damage binding (DDB) and XPC-Rad23B complexes that constantly scan the genome and recognize helix distortions. Upon identification of a damaged site, subsequent repair proteins are then recruited to the damaged DNA to verify presence of DNA damage, excise the damaged DNA surrounding the lesion then fill in the repair patch.A 2002 paper published by Frit et al. showed that Global Genome Repair can be enhanced near transcribed regions. Their main conclusion is that the binding of transcription factors led to an increase in nucleotide excision repair on nearby regions of DNA. They showed that this enhancement was distinct from Transcription Coupled Repair in two ways: Repair was occurring on both the template and non-template strands. Secondly, when Pol-II activity was blocked, either by α-amanitin or modification of the TATA box
TATA box
The TATA box is a DNA sequence found in the promoter region of genes in archaea and eukaryotes; approximately 24% of human genes contain a TATA box within the core promoter....
, the repair still occurred. Through digestions with micrococcal nuclease
Micrococcal nuclease
Micrococcal Nuclease is an endo-exonuclease that preferentially digests single-stranded nucleic acids.The rate of cleavage is 30 times greater at the 5' side of A or T than at G or C and results in the production of mononucleotides and oligonucleotides with terminal 3'-phosphates...
they determined that the binding of transcription factors led to local chromatin
Chromatin
Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
remodeling, likely giving NER proteins greater access to the DNA.
Transcription coupled repair in Eukaryotes
There is a difference in NER efficiency between transcriptionally silent and transcriptionally active regions of the genome. This arises largely because- for many types of lesions- NER repairs the transcribed strands of transcriptionally active genes faster than it repairs the nontranscribed strands and faster than it repairs transcriptionally silent DNA- this particular mode of NER is called TC-NER.At any given time, most of the genome in an organism is not undergoing transcription. The genome-wide process which repairs damage in both transcribed and untranscribed DNA strands in active and inactive genes is called GGR – this process is not dependent on transcription.
TC-NER and GGR are two subpathways of NER, differing only in the initial steps of DNA damage recognition. The principal difference is that TC-NER does not require XPC or DDB proteins for distortion recognition in mammalian cells. TC-NER is instead thought to be initiated when RNA polymerase stalls at a lesion in DNA. In TC-NER the blocked RNA polymerase serves as a damage recognition signal, replacing the need for the distortion recognition properties of the XPC-RAD23B and DDB complexes. Subsequent steps in TC-NER utilize the NER factors XPA, TFIIH, and RPA as well as the nucleases ECC1-XPF and XPG for dual incision at a lesion. The repair patch size for mammalian cells in vivo is around 30 nucleotides, indistinguishable from the repair patch size for GGR.
Excision Enzyme cuts at the Nucleotide excision repair. (L Bridgewater, experiment TAB)