Nucleotide excision repair

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.

Uvr Proteins
The process of nucleotide excision repair is controlled in E. coli 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.

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. XPA, 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.

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, 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.

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)