ABSTRACT

Recent studies identified that CSA belongs to a larger protein complex, which has ubiquitin ligase activity31 and substrate specificity for CSB protein.32 The complex contains the cullin 4A scaffold, R o d R IN G finger and the D N A damage binding protein (D D B 1) adapter that interacts w ith CSA.31 A second, almost identical ubiquitin complex also exists that contains another W D repeat protein, D N A damage binding protein 2 (D D B 2/X PE), in place o f CSA .1330,31 Thus, these two W D repeat proteins likely function as substrate selectors for their complexes that function op­posing N ER sub-pathways. Significandy, the C O P9 signalosome (CSN), a regulator o f cullin-based ubiquitin ligases, may interact w ith the CSA and D D B2 complexes.31 C SN remains bound to the D D B2 complex and inhibits ubiquitin ligase activity, until U V irradiation causes its release. The D D B2 and C U L4A subunits o f the complex are then polyubiquitinated, as are the early stage G G -N ER protein, X P C and chromatin proteins.3133 This activity probably provides access to D N A lesions in regions o f transcriptional inactivity, allowing for assembly o f the G G -N ER machinery. The CSA complex is CSN-free until U V radiation stimulates an interaction,2931 which inhibits the CSA ligase complex activity at the early stages o f T C R . This initial inhibition may perm it the recruitm ent, assembly and repair functions o f the T C R pathway components. A t a later stage the ligase activity o f CSA complex might then allow for removal o f these components when repair is complete and for resumed transcription. Notably, CSB appears to be required for the early T C R stages, before being ubiquitinated by the CSA complex and being subsequendy degraded.32 This observation lead to a hypothesis for why CSB mutations lead to CS,32 while the complete lack o f CSB protein causes U V sensitivity but n o t CS.34 The m utant CSB protein may no t be readily ubiquitinated by CSA and then subsequendy degraded; thus bound CSB interferes w ith later stages in the repair process and inhibits the restart o f transcription. However, potential concerns w ith this hypothesis include that degradation o f CSB does no t coincide w ith the measured linear kinetics o f T C R over a period greater than 30 hr.35 Also, a recent study has described a case o f adult onset o f neurodegeneration in a CS individual w ith a null m utation in the CSB gene.36 An alternate study has provided a second model w ith some differences in functions for CSA and CSB proteins.29 CSB was concluded to act as an initial coupling factor attracting key N E R proteins and the histone acetyltransferase p300, to a stalled R N A P II.29 W hile the role o f CSA complex,

presumably w ith CSB, is to recruit other required proteins likely function in T C R , including H M G N 1, XAB2 and TFIIS, to the stalled RNAP II. Thus, mutations in CSA and CSB likely affect these coupling functions. However, similar to the previous model, the CSA complex also functions to prevent ubiquitination and degradation o f stalled RNAP II and T C R components at a lesion site. Thus, an interesting picture is emerging, where CSA and CSB are required for the assembly o f the T C R machinery and the regulation o f this machinery occurs through ubiquitina­tion. However, further studies are still required to define the precise mechanism o f action o f the CSA ubiquitin ligase complex and its targets in T C R and therefore how mutations in CSA give rise to a CS phenotype. CSB ProteinRecent studies suggest that CSB protein has two key functions, recruiting TCR-pathway pro­teins to the site o f a stalled RNA polymerase and a role in the base excision repair pathway (BER) that repairs oxidative base damage.37 The CSB gene (C K N 2/E R C C 6 ) is located at lO q l l38 and mutations in CSB account for approximately eighty percent o f CS cases.14 CSB encodes a 1493 amino acid protein, which has sequence similarity to the S W I/SN F family o f chromatin remodeling proteins39 and functions as a dimer.40 CSB exhibits DNA-dependent ATPase activity41,42 and the preferred substrate is dsDNA w ith a shorter bubble or loop, mimicking potential D N A structures in N ER and transcriptional arrest43CSB protein has seven conserved ATPase motifs,39 which have been analyzed in homologues by structure-function studies and partially defined in CSB through mutagenesis42,43 (Fig. 2). CSB motifs I and II are classical Walker A and B motifs, respectively, required for ATP-binding and hydrolysis.44,45 N ot surprisingly, mutations in CSB Walker A and B remove ATPase activity and also chromatin remodeling in vitro.42,43 In vivo, Walker A and B mutations perturb recovery o f RNA synthesis after U V exposure, have a failure to complement the UV-sensitivity and increased in apop­tosis.46"48 Similar defects are also observed with mutations motifs la and III, which are likely required for energy transduction from the ATPase site to the oligonucleotide-binding site.44,45,49,50 Studies on m otif IV have not been completed but in homologues it is required for binding dsDNA.44,45 M otif V is expected to bind ssDNA directly and mutations in this m otif and the adjacent m otif VI also have similar T C R defects to mutations in the previously described motifs49,50 and nearly

complete inhibition o f ATPase activity.43 However, mutations in motifs V and VI differ by also having a perturbed reaction to oxidative damage, w ith an increased gamma-irradiation sensitivity and reduced incision o f the most ubiquitous form o f oxidative D N A damage, 8-hydroxy-guanine.49 This may indicate that CSB functions in BER are linked to D N A binding rather than its ATPase activity. Notably, CSB has been observed to control the transcriptional levels o f a fundamental BER protein, O G G I,51 suggesting one potential route for CSB to affect this cellular pathway. Several other motifs are observed in CSB including, an acidic-rich region, a C-term inal Walker A motif, a glycine-rich stretch, a hydrophilic segment and a nuclear localization signal However, mutations in these regions have no t yet been observed to have affect on CSB function.46,49,50,52CSB can alter bo th D N A conform ation on binding and the arrangement o f nucleosomes and also has homologous D N A strand pairing activity.53 This indicates that CSB likely acts as a chromatin-remodeling factor54 to either assist damage repair o f active genes,55 or to aid RNAP I-and II-mediated transcription.56'59 CSB binds to stalled RNAP II and acting as a coupling factor to recruit downstream proteins in the repair pathway.56,60,61 This includes o f the transcription factor T F IIH , which is critically required for both G G -N ER functions and T C R . Key protein-protein interactions may also mediate a potential BER role o f CSB.37 This includes that the efficient repair o f oxidative base lesions occurs through an interaction o f CSB and the BER protein PARP-1 (poly(ADP-ribose) polymerase l) .62,63 CSB also interacts w ith BER protein apurinic/apyrimidinic endonuclease 1 (APE-1) in vivo and in vitro, which stimulates APE-1 activity in an ATP-dependent manner.64 Moreover, CSB may be in a common complex w ith O G G -1, as they colocalize together after y-irradiation.51 However, m uch still remains to be defined for the functions o f CSB (and its regulation by CSA). Further studies will hopefully determine which protein-partner interactions are required for the predom inant roles o f CSB, w hat are the precise mechanisms o f action and how disruption o f the CSB structure leads to CS phenotype. XPB HelicaseThe two helicases associated w ith CS, XPB and XPD, belong to the general transcription factor T F IIH .65,66 Electron microscopy studies revealed that T F IIH forms ring-like structure that has a protrusion on one end and the central cavity o f the ring is large enough to accommodate dsDNA67,68 (Fig. 3a). The ring-core is built from XPB and X PD helicases, together with the protein partners p62, p52, p44, p32 and p 8 /T T D A and is sufficient to support N ER functions o f T F IIH .69,70 The protrusion is the CAK complex o f proteins that are required for phosphorylation o f RNAP II and for transactivation o f nuclear receptor genes.71,72The X P B (ERCC3) gene is located at chromosome 2q2173 and encodes a 782 amino ad d protein that, similar to dose homologues, unwinds D N A with a 3'-5 ' polarity.66,74,75 This helicase activity is essential for prom oter D N A melting and clearance steps during RNAP II initiation o f transcrip­tion and for its repair functions.75,76 The molecular mechanisms o f this dual function have begun to be revealed, through structural and biochemical studies.77'79 The crystal structure o f the archeal Archaeoglohus julgidus XPB (AJXPB) hom olog has been determined79 (Fig. 3b).AfiCPB shares a common core but not the N and C-term ini extensions o f the hum an protein. The^/X PB structure contains two RecA-like helicase domains (H D 1 and H D 2) that belong to helicase superfamily 2 (SNF2), as originally suggested by sequence comparison. However, other unpredicted functional regions in XPB were also discovered. This includes a small damage recognition dom ain (DRD) in the N-term inal region o f H D 1 (Fig. 3b). The D R D shares structural similarity to the mismatch recognition dom ain o f the D N A mismatch repair protein MutS,80 and it also recognizes damaged D N A.79 However, XPB D R D likdy recognizes N ER distortions in the D N A rather than MutS-like lesions, because it is missing the critical Phe residue used in mismatch-specific interactions. Thus, the discovery o f the XPB D R D region may explain how D N A damage is both located and linked to initiation o f D N A unwinding, during N E R steps by XPB helicase. Another discovery was the highly conserved, XPB-family specific, RED amino acid m otif in dom ain H D 1 (Fig. 3b), which m utational analysis suggests has a critical role in D N A unwinding.79 Also observed is a thum b dom ain (ThM ) insert in H D 2, which is predicted to b ind D N A independent o f sequence via the

phosphodiester backbone (Fig. 3b). This is due toA fXPB ThM having similarity D N A polymerase ThM domains and the presence o f several conserved positively charged amino acid residues at the interface between the ThM and H D 2 domains.79XPB likely functions through ATP hydrolysis being coupled to translocation along duplex D N A through large conformational changes in the protein, similar to that indicated for other helicases.81,82 This is supported by the relative orientation o f H D 1 and H D 2 in apo-^/XPB being83 different to the “closed” conformation observed in crystal structures o f nucleotide-bound helicases.84 To close AfX .PB a significant reorientation o f H D 1 and H D 2 would have to take place (Fig. 3c) and these motions likely occur through the long flexible loop that connects the N-and C-term inal helicase

domains. Thus, this structural information provides possible mechanisms for the involvement o f XPB in the unwinding o f duplex D N A during transcription and D N A repair. The interaction o f the XPB ThM and H D 2 domains with 3 '-overhanging D N A may induce a reorientation o f heli-case dom ain H D 2. A nearly 180° change would allow XPB to wrap around the D N A and initiate unwinding. Also, XPB D R D dom ain likely recognizes distorted/dam aged D N A and may induce a similar conformational change during repair. In both situations this closed configuration ideally places the RED m otif at the helicase active site, w ith its side chains intruding into the distorted D N A duplex. This likely allows the RED m otif to function as a molecular wedge, to unzip the D N A when ATP hydrolysis drives XPB along the duplex D N A . However, an unconventional mechanism has also been suggest for D N A melting by XPB; where XPB potentially functions as a molecular “wrench” rotating downstream D N A relative to the fixed upstream protein-D N A interactions.85 This would mean that the AJKPB structure likely represents the transcriptional mode o f XPB, whereas the proposed dom ain reorientation is NER-specific and only occurs upon the interactions o f the D R D with damaged D N A . Significandy, in either proposed mechanism the conformational change o f XPB is critical for switching between the transcription and D N A repair functions o f T F IIH .Hum an m utations exclusively occur in the N-and C-term inal extensions o f hum an XPB, suggesting m utation to the core region is lethal. These hum an XPB extensions may contribute to increased complexity o f interactions and provide a greater control o f function. For example, phosphorylation o f Ser-751 in the C-term inal extension was reported to regulate T F IIH activ­ity in N ER .78 Also, the interactions between XPB and partners, bo th within and outside o f the T F IIH complex, are observed to occur in the XPB extensions and to have profound effects on the T F IIH activities in either transcription or D N A repair.70,77 Therefore, future studies may help define how the structural and mechanistic features o f XPB that are highlighted above fit into the T F IIH complex. This should include how damage recognition signals are propagated and how partner interactions coordinate the functions o f XPB in N E R sub-pathways. XPD HelicaseThe second helicase o f the T F IIH complex, X P D (E R C C 2), is located at chrom osome 19ql3.2-13.3 and encodes a 760 amino acid D N A helicase.86 XPB and X P D proteins are alike in their shared similarity to SNF2 helicases, w ith the key family motifs being conserved in X PD (Fig. 4) and because X P D also functions in both transcription initiation and prom oter escape and in N ER .87'89 However, several key differences exist between XPB and X PD proteins. For example, X PD unwinds dsDN A with the opposite 5'-3' polarity.90 Also, X PD contains a novel iron-sulfur (Fe-S) cluster located between ATPase motifs la and IP 1 (Fig. 4). Furthermore, mutations have been observed throughout the X PD protein and they can cause a third disorder term ed tricho-thiodystrophy (T T D ), in addition to the XP and XP-CS syndromes (Fig. 4). T T D is distinct from X P and XP-CS and its hallmark is sulfur deficient, brittle hair and other symptoms include mental retardation and reduced stature.8The 5'-3 ' directionality o f X PD indicates that its helicase activity is predominantly required to open D N A for repair. In vitro, this helicase activity is relatively weak90 but an interaction w ith its partner protein in T FIIH , p44, increases activity by 10-fold.92 This helicase activity also requires the presence o f the novel Fe-S cluster,91 which is unique to the XPD-like proteins among all character­ized classes o f helicases. This includes the XPD-like FANCJ helicase that causes the cancer-related Fanconi anemia disorder when mutated.. In general, Fe-S clusters are one o f the most abundant and diversely employed enzymatic cofactors.93 However, they are a rarity among nuclear proteins, since D N A glycosylases are the only other Fe-S proteins observed in the nucleus.94,95 The Fe-S clusters are typically utilized for their ability to accept and donate electron, for their tight binding to oxygen and nitrogen atoms and for their ability to stabilize structures. Four Cys residues are conserved in eukaryotic and archeal X PD proteins and these are likely required for Fe binding; m utation in one o f the Cys in the Fe-S cluster removes helicase but no t ATPase activity o f the archeal Sulfolobus acidocaldarius (&zrXPD) X PD homologue.91 Moreover, Saccharomyces cerevisiae strains with a

similar Cys m utation are defective in the N E R o f photoproducts.91 Notably, amino acid R 112 resides in the Fe-S cluster and an R112H mutation is one o f the more frequent causes o f T T D .96 The R112H m utation results in loss o f X PD helicase activity in vitro and defective N ER 72 and a reduction in T F IIH levels in vivo.97 Also, m utation o f the equivalent residue in &*cXPD removes Fe binding and helicase activity,91 Interestingly, a potential function for the Fe-S cluster in X PD has been recently described, which helps explain how the R112H mutation affects protein function and causes the T T D phenotype: Studies on the archeal X P D protein from Ferroplasma acidarmanus have revealed that the intact cluster is critically required for the correct orientation o f the X PD protein at the ssDNA-dsDNA junction, which is required for its unwinding activity.98The three distinct disorders arising from X PD have been suggested to be due to the different transcription verses repair functions o f XPD.99 Initial support for this hypothesis came from an ATPase defective m utant in yeast X PD (Rad3). The Rad3 m utation abolished N E R but the cells were still viable.100 Later studies revealed that human X PD protein was dispensable for transcription initiation in vitro, but its presence substantially stimulates transcriptional activity.88 More specifi­cally, an X P D m utant that lacked ATPase activity was demonstrated to also lack helicase activity, but T F IIH containing this mutation was viable for its transcription functions.101 However, cleavage o f either side o f a D N A lesion was removed by this mutation, indicating that the X PD helicase activity is required in N E R ,101 most likely to allow access for the late-stage endonucleases. This also explains why X PD is relatively tolerant o f mutations w ithout affecting cell viability, since the transcriptional role o f X PD is to maintain stability o f the T F IIH complex. Thus, a current model is that XP and XP-CS are syndromes w ith repair deficiencies, since XP and XP-CS mutations are observed to remove enzymatic activity and inhibit X PD repair functions. T T D instead caused, by mutations possibly affects X PD structural stability and hence transcription.Interestingly, many o f the XP and XP-CS m utations are observed in the C-term inus o f XPD, the site o f interaction with the p44 partner that stimulates its activity89 (Fig. 4). This gives further support to the hypothesis for XP and XP-CS being repair specific syndromes. However, higher resolution structural analyses would be useful in complementing the electron microscopy structure o f T F IIH , to provide a much clearer understanding o f m utational deficiencies. New structural

information may explain how the X PD mutations are able to cause the different phenotypes, even when the m utations occur in adjacent am ino acids. For example, the G602D m utation results in XP-CS, while the neighboring R 601L /W m utation instead causes XP. XPG EndonucleaseThe X P G (ERCC5) gene was located at chromosome position 13q32.2-q33.1102 and the protein product was observed to correct cells from individuals showing deficiency o f X P G complemen­tation group, which are highly U V sensitive and NER-deficient.103,104 Studies on X PG protein revealed that it is an 1186 residue containing structure-specific nuclease.105'107 X PG functions in the late stage o f N E R by incising the damaged D N A strand in a transcription bubble structure, zero to two nucleotides from the ssDNA-dsDNA junction.108 O ther roles for X PG have also been indicated, which include transcription109 and the repair o f oxidative base damage.110,111 X PG protein is a member the FEN -1 (Flap EndoNuclease-1) family o f structure-specific nucleases (Fig. 5). M utations that perturb the nuclease function o f the protein cause an XP phenotype, while others that truncate the protein cause X P-C S112'114 (Fig. 5).The structure o f X PG protein is undetermined, but structures o f family members are observed to form an a / (3 saddle* structure built from two regions, N and I, which are separated by a flexible loop o f approximately 70 amino acids.115118 The X PG sequence differs from the family by containing a large spacer region o f over 600 amino acids (the Recognition, R-domain) and a C-term inus that is extended by approximately 145 residues (Fig. 5). A seven-residue deletion in the R-domain and a truncation o f260 residues in the C-term inus can give rise to the XP-CS phenotype.112,114,119 This indicates that both nonenzymatic regions have critical functions in the cell The large R-domain sequence does not contain clear sequence similarity to any known structural motifs, but it is required for X PG bind to N ER bubble substrates.120’122 XPG bound on bubble substrate interacts with CSB, which stimulates both bubble D N A binding and ATPase activity o f CSB and this interaction is

mediated by the X PG C-term inus extension.122 This finding implicates X PG function in the early recognition stage o f T C R that requires CSB protein. This early stage function is also suggested by interaction o f X PG with elongating R N A P II in vivo and with stalled R N A P II in vitro122 Stalled RNAP II blocks the X PG incision activity in vitro but this can be removed by the addition o f T F IIH and ATP.122 Notably, X PG has also been observed to bind T F IIH .123 M utants o f XPG protein lacking the C-term inus due to XP-CS mutations did not form a stable complex with T F IIH , while X PG m utant proteins with the classical XP phenotype bind efficiently.123 Altered X PG protein that failed to associate with T F IIH also caused this dissociation o f CAK subunit from the T F IIH core.123 SiRNA knock down o f X PG results in CAK dissociation, while expression o f wild-type X PG restores the interactions and T F IIH architecture.123 Overall, this indicates that the XP-CS phenotype might arise from perturbed interactions with either the CSB protein partner and /or with T F IH in m utant forms o f XPG. Moreover, T F IIH is required for transactivation o f nuclear receptors and the loss o f this activity might cause some o f the phenotypes o f XP-CS in XPG. This would be similar to XP-CS mutations in X PD, likely altering T F IIH architecture and resulting in the loss o f subcutaneous fat tissue and hypogonadism.71,124*125 However, loss o f T F IIH transactivation might no t common to all CS-linked mutations, since transactivation is normal in X P-CS o f CSA, CSB and XPB mutants. Thus, further studies to define the common transcription/repair deficiencies among the CS-linked proteins are needed. Perspectives

We have presented the recent findings on the structures and protein partner interactions o f the five proteins mutations in which cause either CS or CS syndrome that is combined with XP. These novel studies are providing a considerable increase in our understanding o f the molecular basis o f the complex disease phenotype o f CS. This also includes results defining catalytic mechanisms which allows for comparison with the molecular mechanisms o f other key D N A repair proteins, such as R ecQ helicases that cause progeria when defective or the M rell-R ad50-N bsl (M RN) complex that can cause neurological disorders when mutated, to define common functionalities and key pathway differences.126 These types o f studies also indicate that the CS-linked proteins and their key complexes are regulating T C R pathway progression. This occurs through protein conformational changes, induced by signaling events that may include ubiquitination and by binding o f substrate or protein partners. Thus, continuation o f these studies will likely provide a detailed understanding o f how m utation o f specific gene results in the CS phenotype. This is particularly the case w ith the XPB, X PD and X PG proteins: the combination o f high resolution protein crystallography and lower resolution small angle X-ray scattering studies127,128 will likely define how CS mutations uncouple enzymatic activities and perturb conformational changes and/ or protein partner interactions. However, molecular studies on CSB and probably CSA will also have to include new analyses o f genetic and /or environmental factors. This is because the same inactivating m utation in CSB has been observed to affect XP and/or CS phenotypes.129A current model for CS is that neurodegeneration may be caused by an apoptotic response to lesions blocking transcription. The brain, w ith its lim ited capacity for cellular proliferation, therefore undergoes a loss o f cells leading to the CS phenotype. However, alternate models for the CS phenotypes also warrant further investigation. O ne group has suggested that the CS phe­notype may be due deregulation o f additional downstream targets o f the CSA E3 ubiquitination system.130 Supporting this is their observation o f increased protein levels in CS cells, including p21 protein that is known to result reduced repair and increased oxidative damage.130 A second group has reported breaks in the D N A o f X P D cells with XP-CS mutation that are no t localized at sites o f D N A damage;83,131 instead, they probably occur at sites o f transcription initiation and thus may be partially responsible for XP-CS phenotype specifically in XPD.Interestingly, the increased apoptosis in CS cells might be reason behind the lack o f tumorigen-esis in CS individual, despite increased rates o f point mutations in their D N A. This feature, along with the sensitivity o f N E R deficient cells to interstrand cross-linkers that are used as therapeutic against cancer, suggests the CS proteins and their partners in D N A repair pathway progression may