Human genes (and the corresponding proteins) have been identified by their ability to correct the repair defects in cells from individuals with NER deficient syndromes [6, 7].  Other human repair genes were identified by the ability to correct repair deficient chinese hamster ovary (CHO) cell lines [8, 9], giving rise to the excision repair cross complementing (ERCC) group of genes.  Many of the ERCC genes were subsequently found to be identical to the XP and CS genes.  Some genes were also isolated by homology to previously identified rad group of yeast repair genes, which was facilitated by the high degree of conservation of eukaryotic NER genes (Table III).

      Of the identified human NER proteins, at least four have been implicated the process of damage recognition.  These proteins appear to segregate into two complexes: 

- The XPA [10, 11], XPE [12-14] and RPA [15-17] proteins can each bind damaged DNA independently, but interactions between them further increase this affinity [17-20]. 

- The second complex with a strong affinity for damaged DNA [21, 22] consists of the XPC protein in a tight association with a human homologue of the yeast rad 23 (HHR23B) protein.

      Like the prokaryotic model, it is likely that lesion recognition involves multiple steps, with the requirement for  identification of damage by one complex to be verified by the other before the repair complex is assembled.  However there is controversy regarding the order in which the NER proteins are assembled on UV photoproducts.   It is even possible that the order of assembly may vary depending on the specific nature of the lesion (see figure below). 

 The XPC protein has an affinity for both single-stranded [21, 23] and double-stranded DNA [21], and can bind a variety of DNA damage [22].  XPC exists in a tight complex with HHR23B [24] and is the first NER protein to recognise NA-AAF induced DNA damage [22].  XPC/HHR23B also associates with TFIIH [25].  Both XPC and TFIIH are required to form an open complex around a cis-diammine-dichloro-platinum (cis-platin) adduct allowing the subsequent addition of XP-A and RPA [26].  The formation of an open complex is presumably mediated by the helicase activities of the XPB and XPD components of TFIIH [25, 27-30], which then recruits XPA through a direct interaction [31].  Interestingly, the yeast homologue of HHR23B (Rad23) has been demonstrated to increase the interaction between TFIIH and Rad 14 (the yeast homologue of XPA) [32], although the effect of HHR23B on human NER remains controversial [21, 23].  The addition of XPA and RPA (through its interaction with XPA [17, 19, 33]) stabilises XPC/HHR23B and TFIIH on the damaged DNA to form pre-incision complex 1 (PIC1) [34, 35]. 

     Although the XPC/HHR23B complex appears to be the first to recognise several different forms of DNA damage [22, 26], it has been proposed that a second complex comprised of XPA, RPA, XPE and XPF/ERCC1 plays a significant role in the recognition of 6-4 PPs.  The XPA, XPE and RPA proteins all have a very strong, specific affinity for 6-4PPs [10, 12, 14, 15].  Whereas, neither XPA nor RPA nor a combination of the two is capable of discriminating between CPD containing DNA and undamaged DNA  [34].  A strong interaction between XPA and ERCC1 [36-39] further increases the affinity of XPA for UV damaged DNA [39], and cells from patients with mutations in the XPF gene exhibit deficient 6-4 PP repair but normal CPD repair [40-43] (see also Dual roles for ERCC1/XPF ?).  Both XPA [17, 19, 33] and XPE [18] directly interact with RPA, and these interactions further increase the binding affinity of these proteins for UV-damaged DNA [17-19].  These data suggest the existence of a large multi-subunit complex with a strong, specific affinity for 6-4PP.

     RPA is a ssDNA binding protein, which binds to the undamaged strand during NER [44] and can unwind short dsDNA substrates [45-47].  As 6-4 PPs strongly favour disruption of the DNA double helix [48], this distortion may be sufficient for RPA to further unwind the DNA allowing lesion identification by XPA (and/or the remainder of the 6-4 PP recognition complex).  Although XPA and TFIIH do not interact well in the absence of DNA damage, the binding of XPA to damaged DNA results in a conformational change which greatly increases its affinity for TFIIH [31, 49].  Recruitment of TFIIH is expected to also bring in XPC/HHR23, which appears to be necessary to promote the stable binding of XPA on UV irradiated DNA [34, 35, 50].  At the same time, it is likely that ERCC1/XPF disassociates from XPA, as it is not associated with the first stable protein complex on UV-damaged DNA [34, 35].  Consistent with this model, a kinetic analysis has shown that a complex of XPA and RPA binds a 6-4 PP adduct prior to XPC/HHR23B [51].  As XPE is not required for NER in vitro [52], its association with the stable recognition complex has not been examined.
 
 

     The first stable complex on damaged DNA (pre-incision complex 1 or PIC 1) consists of XPA, RPA, XPC/HHR23B and TFIIH [34, 35] (see figure above).  RPA binding to ssDNA protects ~30 nucleotides [53-55] and appears to define the size of the excision fragment in NER [44, 56]. 

     Addition of the 3' endonuclease [57], XPG, results in the concomitant loss of XPC from the complex and yields in PIC2 [35].  As RPA binds the undamaged DNA strand with a defined polarity [44], its interaction with XPG [17], specifically positions XPG at the 3' end of the fragment to be excised [44, 58].  However, an interaction between XPG and TFIIH [59], may also play a role in its recruitment to the repair complex.

     Finally, the XPF/ERCC1 complex is recruited to form PIC3 [35].  A direct interaction between RPA and XPF [60] positions XPF/ERCC1 at the 5' end of the fragment to be excised [44, 58].  The strong XPA-ERCC1 interaction does not appear to play a significant role in this positioning [58].  The interaction of RPA with XPG and XPF/ERCC1 stimulates the junction cutting activities of each of these structure specific endonucleases [44, 58, 60].  XPG incises the damaged strand at the 3' end of the open complex, followed closely by an incision at the 5' end of the complex mediated by XPF/ERCC1 [57, 61, 62].  Although there is some variability in the exact incision sites, the cuts are made at approximately 6 phosphodiester bonds 3' and 22 phosphodiester bonds 5' of the lesion, yielding an excision fragment of 27-29 nt [56].

     Unlike the prokaryotic model, in eukaryotic NER the dual incisions have been reported to result in excision and release of the damage containing oligonucleotide without the aid of additional repair factors [61], perhaps as a result of the helicase activities of XPB and XPD.  Resynthesis of the excised fragment is mediated by DNA pol d or e [63], and sealed by DNA ligase I [64].
 

Transcription-coupled repair in Eukaryotes
 

     Damage in the transcribed strand of active genes is believed to be recognised by its ability to block transcription complexes.  The CSB protein is a component of the RNA pol II elongation complex [65-68], and is believed responsible for recruiting TFIIH to stalled RNA pol II complexes [68-70].  The interaction between CSB and TFIIH is increased by the presence of CSA [68].  TFIIH-mediated unwinding of the DNA around the stalled RNA pol II complex may facilitate elongation past intrinsic pause sites, but is also likely to function in the recruitment of the NER complex when RNA pol II is blocked by DNA damage.  As eukaryotic genes are significantly larger than their prokaryotic counterparts, it is believed that the increased metabolic cost of aborting and reinitiating transcripts in eukaryotic cells has resulted in a mechanism which permits a stalled RNA pol complex to resume mRNA synthesis once the transcription blocking lesion has been removed.  Consistent with this model CSB, unlike the prokaryotic TRCF, does not dissociate the stalled RNA pol II complex from the DNA [71], furthermore a stalled RNA pol complex does not inhibit in vitro repair of the transcription blocking lesion [72]. 

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