The NER system recognizes and repairs DNA damage that consists of ultraviolet (UV)-induced photoproducts and large DNA adducts. These include the cyclobutane [5-5], [6-6] pyrimidine dimers and the [6-4] pyrimidine pyrimidinone dimers that can involve T and C pyrimidines. Chemical adducts include those produced by AAAF, benzo(a) pyrine, photoactivated psoralens, cis-platinum and many others. Most Xray damage is processed differently using single strand and double strand break repair pathways; most single base damage caused by alkylating agents or oxidative reactions are repaired by base excision repair and alkyltransferase. The distinction is not absolute, however, and there may be some overlap in the substrate specificity of these various repair systems.

Two major branches of NER are distinguished by their relationship to transcriptional activity of the genes being repaired. These are described as transcription-coupled repair (TCR) and global genome repair (GGR). CPDs are excised more rapidly from actively transcribed genes, especially from the DNA strand used as the template for transcription (Mellon et al. 1986). This strand preference also requires the action of components of the mis-match repair (MMR) system whose major role is correcting mis-matched bases generated through replication errors or deamination of cytosine, and can discriminate parental from newly synthesized DNA strands (Leadon and Avrutskaya 1997; Mellon et al. 1986). In addition, a basal transcription factor, TFIIH, plays a major role in repair and many of its components are directly involved in remodeling the damaged regions for excision to occur (Schaeffer et al. 1993). The initial damage recognition mechanism for TCR may be the stalled RNA pol II, itself.

The initial damage recognition factors uniquely required for GGR are the XPC and XPE DNA binding proteins. The XPC-hHR23B complex (Masutani et al. 1994; Shivji et al. 1994) is the earliest damage detector to initiate NER in nontranscribed DNA, acting before the XPA protein, and serves to stabilize XPA binding to the damaged site with a high affinity for the [6-4]PP (Sugasawa et al. 1998; Wood 1999). The XPC protein may be required for transient nucleosome unfolding during NER (Baxter and Smerdon 1998). This complex is specifically involved in GGR but not TCR. Stable association of TFIIH with DNA lesions is dependent on the integrity of XPA and XPC proteins. The XPE protein has similar binding characteristics to photoproducts as XPA and XPC but plays a much less prominent role. XPE is a heterodimer of a p48 which is found to carry mutations from several XPE patients, and a p125 protein (Hwang et al. 1998). The p48 subunit is inducible in human cells in a p53-dependent manner and is not expressed in hamster cells that fail to repair CPDs in nontranscribed DNA (Hwang et al. 1999). An in vitro study of the binding activities of various NER components, however, suggests that the initial binding factor on DNA damage, may be the XPA-RPA complex because it has the highest binding constant (Wakasugi and Sancar 1999).

2. Unwinding and formation of a repair complex

Unwinding of the damaged site involves the transcription factor TFIIH that contains both 3'-5' (XPB) and 5'-3' (XPD) helicases. CSA and CSB proteins are involved in coupling the RNA pol II transcription arrest at photoproduct sites to the unwinding events. The unwound region is stabilized by the XPA binding protein and the RPA heterotrimeric protein that binds to both XPA and DNA.

The individual factors of NER associate sequentially and independently on UV photoproducts, to a first approximation without preassembly of a "repairosome" complex (Houtsmuller et al. 1999; Volker M et al. 2001). The basic components of the process include the XPA protein, the heterotrimeric replication protein (RPA), the 6 to 9 subunit TFIIH, the XPC-hHR23B complex, the XPG nuclease, and the ERCC1-XPF nuclease (Sancar 1994). The final stable complex that remains at the damaged site is unclear but is a subset of these components that act as an assembly point for the nucleases that cut the damaged strand.

The excision process involves removal of a 27-29 nt oligonucleotide containing the photoproduct by cleavages 5 nt on the 3' side of the photoproduct, and 24 nt on the 5' side (Huang and Sancar 1994). Slight variation in the precise sites of cleavage result in the removal of 27-29 nt. The XPG nuclease cleaves on the 3’ side of the damage; the XPF-ERCC1 heterodimer cleaves on the 5’ side of the damaged site. The XPF and ERCC1 genes may represent an ancient duplication and are unstable unless in a heterodimer complex (Gaillard and Wood 2001). XPA serves as an anchor for the 5’ nuclease through binding to ERCC1 (Saijo et al. 1996).

The excised region is replaced by the action of a complex similar to that involved in normal DNA replication. PCNA is loaded onto the DNA by the 5 subunit RFC complex which then anchors replicative polymerases Pol D or Pol E.

The final closure of the repaired site occurs with DNA ligase, most probably ligase I

7. Replication of damaged DNA

DNA photoproducts are blocks to the replicative DNA polymerases, alpha, delta and epsilon (Pol A, D, E) which cannot accommodate large distortions such as DNA photoproducts or adducts in their active sites (Brash et al. 1991; Steitz 1999). Replicative bypass of these photoproducts is achieved instead by damage-specific polymerases with relaxed substrate specificity, now defined as class Y polymerases (Cleaver 1999; Ohmori et al. 2001). Three members of class Y have been identified in the mammalian genome, POL H, I, and K. POL H and I are close homologs, unique to mammalian cells, and only a single POL H gene is found in yeast (Ohmori et al. 2001). Pol I has a poorer capacity for replication of UV damage and Pol K seems completely unable to replicate UV damage. These polymerases have larger active sites that allow them to read-through noninformative sequence information resulting from DNA damage (Trincao et al. 2001). The consequence is that these polymerases have high error rates of the order of 1% when assayed in vitro, and this property must be controlled in vivo otherwise the results would be catastrophic to the cell (Johnson et al. 2000; Matsuda et al. 2000). Control is achieved by several mechanisms for Pol H. First, the enzyme is excluded from the replication fork until replication is stalled by UV damage, at which point Pol H accumulates in foci at the replication fork (Kannouche et al. 2001; Thakur et al. 2001). This requires specific sequence motifs in the protein for translocation and for binding to PCNA (Haracska et al. 2001; Kannouche et al. 2001). Pol H acts distributively, and is only able to extend the nascent DNA chain by one or two bases across from the photoproducts, and there may be a role for editing by a separate exonuclease. This results in the addition of adenines across from thymine-containing photoproducts resulting in accurate replication of a T-T pyrimidine dimer. Subsequent extension of the DNA involves Pol Z which can initiate replication from mismatched 3' termini.