Nucleotide Excision Repair (NER)

NER is a multistep process that recognizes and eliminates a wide spectrum of damage causing significant distortions in DNA structure, such as UV-induced damage, ionizing irradiation, electrophilc chemical mutagens, bulky chemical adducts etc. In higher eukaryotic cells, NER excises 24-32-nt DNA fragments containing a damaged link with extreme accuracy. Reparative synthesis using an undamaged strand as a template, followed by ligation of single-strand break emerging as a result of the damage, is the final stage of DNA repair. For many types of lesions, NER repairs the transcribed strands of transcriptionally active genes faster than it repairs nontranscribed strands and transcriptionally silent DNA.

Two modes of NER can be distinguished: repair of lesions over the entire genome, referred to as global genome NER (GG–NER), and repair of transcription-blocking lesions present in transcribed DNA strands, hence called transcription-coupled NER (TC–NER). Distortions in NER activity can result in UV-sensitive and high-carcinogenic pathologies like Xeroderma pigmentosum (XP), Cockayne Syndrome (CS), and Trichothiodystrophy (TTD), as well as other neurodegenerative manifestations. Currently available information on the main genes inactivated in NER-defective cells and on the protein factors and enzymes encoded by these genes indicates that the process involves the coordinated action of of approx. 30 proteins that succesively form complexes with variable compositions on the DNA.

Studies on NER key factors

In the last three decades, all key NER factors have been cloned and the core of the ‘cut-and-paste’ reaction has been reconstituted in vitro from purified components. We found out that XPC (complexed to hHR23B) is a DNA-damage sensor and repair-recruitment factor; the general transcription factor complex TFIIH, containing the XPB and XPD helicases, mediate strand separation at the site of the lesion; and XPA verifies the damage in an open DNA conformation and is crucial in the assembly of the remainder of the repair machinery. Replication protein A (RPA) stabilizes the opened DNA complex and is involved in positioning the XPG and ERCC1–XPF endonucleases responsible for the DNA incisions around the lesion. After removal of the damage-containing oligonucleotide, typically 24–32 nucleotides in length, general replication factors fill in the remaining gap and close it.

• For the evidence of XPC–hHR23B, DNase I footprinting showed that XPC–hHR23B binds directly to DNA damage and changes the DNA conformation around the lesion. So, XPC–hHR23B initiates GG–NER by sensing and binding lesions, locally distorting the DNA double helix and recruiting the other factors of the system.
• For TFIIH, a number of interactions with repair factors have been reported, including XPA and the aforementioned interactions with XPC. Direct in vitro interactions were also described between multiple subunits of TFIIH and XPG (Iyer et al.1996), consistent with the reported detection of XPG in partially purified TFIIH fractions. A mutant in the carboxy-terminal domain of XPB was shown to fully support DNA unwinding and allow 3′ but not 5′ incision, suggesting that TFIIH facilitates the 5′ incision by ERCC1–XPF. In vitro binding was also described between TFIIH and CSA, an interaction perhaps relevant for TCR. Clearly, in addition to unwinding, TFIIH has other engagements in NER.
• In the case of XPA, it has long been considered the damage-sensing and repair-recruitment factor of NER. However, as XPC–hHR23B was shown recently to act first, the function of XPA has to be reconsidered. Given its affinity for damaged DNA and its ability to interact with many (core) repair factors, XPA is anticipated to verify NER lesions and to play a central role in positioning the repair machinery correctly around the injury.
• For RPA, on the basis of its dual involvement in replication and repair, it was anticipated that RPA not only acts at preincision stages but also during DNA repair synthesis. DNA polymerase δ and ε (Pol δ and Pol ε) have been implicated in repair synthesis, and both can be stimulated by RPA. However, genetic evidence in yeast suggests that RPA and Pol δ do have a direct interaction, and possibly, RPA remains bound to the undamaged strand after excision, thereby facilitating gap-filling DNA repair synthesis, which initiates at the 5′ incision site.
• XPG is not only required for the 3′ incision but also for full open-complex formation, indicating a structural role in the core NER reaction. Evidence for such a role was provided with the D812A active-site mutant of XPG, which had to be present to detect ERCC1–XPF-mediated 5′ incisions in an in vitro-reconstituted repair assay with purified factors. Furthermore, this same XPG mutant was found to stabilize a preincision complex containing XPC–hHR23B, TFIIH, XPA, and RPA. Apparently, independent of its cleavage activity, XPG has a structural function in the assembly of the NER DNA–protein complex. The reported interactions of XPG with TFIIH and RPA may be relevant in this respect. Therefore, XPG, like TFIIH, is anticipated to be a key factor in coupling various repair processes to transcription.
• In ERCC1-XPF, several protein interactions have been reported, that may account for its positioning during NER. XPA interacts with the complex, mainly via ERCC1, although a weak affinity for XPF also has been reported. RPA and ERCC1 likely bind sequentially to XPA. RPA also interacts with ERCC1–XPF, presumably via XPF. This interaction seems particularly important for positioning the nuclease. Bound to ssDNA, the 3′ oriented side of RPA interacts with ERCC1–XPF and strongly stimulates its nuclease activity, whereas the 5′ oriented side of RPA does not interact with the complex and blocks ERCC1–XPF-mediated incisions.
• In case of XPE, some patients carry a mutation in the gene for the small subunit, which is under damage-inducible control by p53. This explains the partial defect in GG–NER in p53−/− cells, supporting the idea that DDB facilitates the identification of lesions that are poorly recognized by the XPC–hHR23B complex, such as UV-induced CPD dimers.

Diseases associated

Popular Citations

• Marteijn JA, Lans H, Vermeulen W, Hoeijmakers JHJ (2014). "Understanding nucleotide excision repair and its roles in cancer and ageing." Nat. Rev. Mol. Cell Biology.
• Aboussekhra A, Biggerstaff M, Shivji MKK, Vilpo JA, Moncollin V, Podust VN, Protic M, Hubscher U, Egly JM, Wood RD (1995). "Mammalian DNA nucleotide excision repair reconstituted with purified components." Cell.