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See online version for legend and references.754 Cell 140, March 5, 2010 ©2010 Elsevier Inc. DOI 10.1016/j.cell.2010.02.033

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SnapShot: Nucleotide Excision RepairCaixia Guo,1,3 Tie-Shan Tang,2 and Errol C. Friedberg3

1Beijing Institute of Genomics, 2SKLBMB, Institute of Zoology, CAS, 100101 Beijing, China; 3UT Southwestern Medical Center, Dallas, TX 75390, USA

754.e1 Cell 140, March 5, 2010 ©2010 Elsevier Inc. DOI 10.1016/j.cell.2010.02.033

Nucleotide excision repair (NER) was discovered in both prokaryotes and eukaryotes in the 1960s (Friedberg et al., 2005). The process corrects a wide spectrum of damage to DNA bases that results in distortions in the native conformation of DNA, including damage induced by ultraviolet (UV) light and by a plethora of chemicals. NER comprises two distinct subpathways. Global genome repair (GGR) repairs lesions in regions of the genome that are transcriptionally silent, and transcription-coupled repair (TCR) repairs lesions in regions of the genome that are transcriptionally active. A key difference between these two NER pathways is the molecular mechanism used to recognize the damaged base (designated by a red star in the SnapShot figure).

NER in ProkaryotesIn prokaryotic organisms, such as Escherichia coli, NER involves three proteins, UvrA, UvrB, and UvrC, which are collectively responsible for both the recognition of a damaged base and its excision from the genome (Friedberg et al., 2005; Truglio et al., 2006). ATP binding and hydrolysis are required for the UvrABC system to function. ATP drives the formation of the UvrA dimer, which possesses ATP/GTPase activity and can interact with UvrB. During GGR, UvrA and UvrB proteins form an ATP-dependent heterotrimer that directly recognizes damaged DNA, whereas during TCR, the Mfd (mutation frequency decline) protein, also called the transcription repair coupling factor (TRCF), recruits UvrA to sites of damaged DNA associated with stalled RNA polymerase (Selby and Sancar, 1993). In both pathways, the UvrA dimer loads UvrB onto damaged DNA. UvrA in the UvrA2B complex initiates contact with DNA and then facilitates the association of the UvrB DNA-binding domain with the damaged DNA. Once the correct UvrB-DNA complex is formed, ATP hydrolysis by UvrA is triggered, and this protein dissociates from the complex, leaving a stable UvrB-DNA preincision complex. The recruitment of UvrC to the preincision complex by UvrB (which requires ATP hydrolysis by UvrB) results in the generation of an endonuclease that incises (nicks) the damaged strand on either side of the lesion. The first incision occurs ?4 nucleotides 3′ of the damaged base, and the second incision occurs ?7 nucleotides 5′ of the damaged site. UvrD protein (DNA helicase II) facilitates the release (excision) of the damaged oligonucleotide (?12–13 nucleotides in length) and dissociation of UvrC from the fragment. DNA polymerase I fills the gap generated in the DNA duplex and triggers the release of UvrB. Newly synthesized DNA is then joined to the extant DNA by DNA ligase.

NER in EukaryotesThe fundamental mechanism of NER is well conserved in eukaryotes, and most components and features of the reaction are very similar in the budding yeast Saccharomyces cerevisiae and in mammals (Friedberg et al., 2005). However, the precise details of NER in higher organisms, especially the order in which components associate on the DNA, are not fully understood. Seven genetic complementation groups of the human disease xeroderma pigmentosum (XP), designated XPA to XPG, are known to be required for NER in mammalian cells. Two independent complexes, one involving the XPC/HR23B/Centrin 2 proteins and the other involving the DDB1/DDB2 heterodimer, have been implicated in the early steps of base damage recognition during NER (Nouspikel, 2009). However, it is not known whether both complexes are always involved in the recognition of all types of damage that are processed by NER.

UV-damaged DNA-binding protein (UV-DDB) is a heterodimeric complex composed of DDB1 and DDB2. Binding of UV-DDB to sites of damage caused by UV radiation activates a UV-DDB-associated ubiquitin ligase complex that recruits XPC protein to the lesion and promotes ubiquitination of DDB2 and XPC proteins (Sugasawa et al., 2005). Polyubiquitinated DDB2 protein loses its ability to bind to UV-irradiated DNA, whereas ubiquitinated XPC shows enhanced binding to DNA.

A multiprotein transcription/repair complex, designated transcription factor IIH (TFIIH), is believed to be recruited to sites of base damage via interaction with XPC/HR23B/Centrin 2, and the unwinding of the DNA helix around such sites is initiated by the XPB and/or XPD ATPase/helicase components of TFIIH (Volker et al., 2001). Subsequent recruit-ment of XPA and replication protein A (RPA) drives detachment of the cyclin-dependent kinase-activating kinase (CAK) subcomplex of TFIIH, which is essential for genome-wide transcription by RNA polymerase II (RNAP II). This leads to unwinding of duplex DNA in a limited area surrounding the damaged base(s) (bubble formation) and the release of XPC/HR23B/Centrin 2 (Coin et al., 2008). XPG is also required for complete bubble formation via its interaction with and stabilization of TFIIH (Coin et al., 2008). XPG and the ERCC1/XPF complex are both structure-specific endonucleases that cut the damaged strand of DNA 3′ and 5′ to the lesion, respectively. This produces a single-stranded oligonucle-otide ?24–32 nucleotides in length. The incisions are asymmetrical, such that the 3′ incision occurs 2 to 8 nucleotides from the damaged base and the 5′ incision occurs 15 to 24 nucleotides from the damaged base. The resulting gap is then filled in by the combined actions of DNA polymerase δ or ε, proliferating cell nuclear antigen (PCNA), RPA, and DNA ligase I (or a complex of XRCC1and DNA ligase III).

Damage recognition during TCR in eukaryotes is believed to be initiated by the stalling of RNAP II at lesions in the transcribed strand (TS). The stalled RNAP II complex recruits CSB protein, which is an ATP-dependent chromatin-remodeling factor in the SWI/SNF family (Hanawalt and Spivak, 2008; Svejstrup, 2002). CSB, in turn, is believed to recruit a number of additional factors to the stalled RNAP II complex, including the histone acetyltransferase p300, the CSA-DDB1 E3 ubiquitin ligase/COP9 signalosome complex (Fousteri et al., 2006), TFIIH, RPA, and other core NER proteins (excluding XPC/HR23B/Centrin 2). In cooperation with CSB, CSA protein may facilitate access to the damaged site while also aiding the association of the stalled transcription complex with multiple factors, such as HMGN1 (high-mobility group N nonhistone nucleosome-binding protein), XAB2, and TFIIS. However, the order of these molecular events and their requirement in the excision repair of specific types of DNA lesions is poorly understood.

Subsequent steps involve the binding of TFIIH and XPA/RPA, to which the incision nucleases XPG and XPF/ERCC1 bind, promoting 3′ and 5′ cleavages of the damaged DNA strand, respectively. As in GGR, the oligonucleotide, thus generated, is removed, and the gap in the damaged strand of the DNA duplex is filled in by the combined actions of DNA polymerases δ or ε, PCNA, RPA, and DNA ligase I or XRCC1/ DNA ligase III.

Acknowledgments

T.-S.T. is supported by grants KSCX2-YW-R-148 (Knowledge Innovation Program of CAS) and NSFC30970931. E.C.F. is supported by grant ES11344 (NIEHS).

RefeRences

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Fousteri, M., Vermeulen, W., van Zeeland, A.A., and Mullenders, L.H. (2006). Cockayne syndrome A and B proteins differentially regulate recruitment of chromatin remodeling and repair factors to stalled RNA polymerase II in vivo. Mol. Cell 23, 471–482.

Friedberg, E.C., Walker, G.C., Siede, W., Wood, R.D., Schultz, R.A., and Ellenberger, T. (2005). DNA Repair and Mutagenesis (Washington, DC: American Society for Microbiology).

Hanawalt, P.C., and Spivak, G. (2008). Transcription-coupled DNA repair: two decades of progress and surprises. Nat. Rev. Mol. Cell Biol. 9, 958–970.

Nouspikel, T. (2009). DNA repair in mammalian cells: Nucleotide excision repair: variations on versatility. Cell. Mol. Life Sci. 66, 994–1009.

Selby, C.P., and Sancar, A. (1993). Molecular mechanism of transcription-repair coupling. Science 260, 53–58.

Sugasawa, K., Okuda, Y., Saijo, M., Nishi, R., Matsuda, N., Chu, G., Mori, T., Iwai, S., Tanaka, K., and Hanaoka, F. (2005). UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin ligase complex. Cell 121, 387–400.

Svejstrup, J.Q. (2002). Mechanisms of transcription-coupled DNA repair. Nat. Rev. Mol. Cell Biol. 3, 21–29.

Truglio, J.J., Croteau, D.L., Van Houten, B., and Kisker, C. (2006). Prokaryotic nucleotide excision repair: the UvrABC system. Chem. Rev. 106, 233–252.

Volker, M., Mone, M.J., Karmakar, P., van Hoffen, A., Schul, W., Vermeulen, W., Hoeijmakers, J.H., van Driel, R., van Zeeland, A.A., and Mullenders, L.H. (2001). Sequential assembly of the nucleotide excision repair factors in vivo. Mol. Cell 8, 213–224.