Nucleotide excision repair

All living organisms are equipped with DNA repair systems that can copewith a wide variety of DNA lesions. Among these repair pathways,nucleotide excision repair (NER) is a versatile repair pathway, involved inthe removal of a variety of bulky DNA lesions such as UV inducedcyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidonephotoproducts (6-4PP). NER is a complex process in which basically thefollowing steps can be distinguished:

• (i) recognition of a DNA lesion;
• (ii) separation of the double helix at the DNA lesion site;
• (iii) single strand incision at both sides of the lesion;
• (iv) excision of the lesion-containing single stranded DNA fragment;
• (v) DNA repair synthesis to replace the gap and
• (vi) ligation of the remaining single stranded nick.

The importance of NER for human health is illustrated by the occurrence ofrare autosomal recessive disorder xeroderma pigmentosum (XP). Patients characteristically show severe photosensitivity andabnormal pigmentation, often accompanied by mental retardation, and theyusually develop skin cancer at very young age (Bootsma et al., 1998) .Cells from these patients are also extremely sensitive to UV light and havea defect in NER. Complementation studies revealed that eight genes areinvolved in XP: XPA through XPG and XPV (XP-Variant). Mutations in the XPgenes (except XP-variant) lead to defective NER and hypersensitivity to UV.The XP variant cells are proficient in NER but deficient in lesion bypasswhen the replication fork encountered a bulky adduct. Normally, thetranslesion synthesis is carried out by the polymerase eta, which ismutated in the XP variant. XP-V patients are more UV-sensitive than normalindividuals but less than classical XP. They develop skin cancers aroundthe age of 20-30 and exhibit less neurological abnormalities.

In addition to XP, other UV sensitive syndromes exist. Cockayne' syndrome (CS) is a rare disorder that is associated with a widevariety of clinical symptoms. Beside other symptoms, the patients generallyshow dwarfism, mental retardation and photosensitivity. In contrast to XP,CS is not associated with an enhanced incidence of skin cancer. Cells fromCS patients are hypersensitive to the cytotoxic effects of UV and arecharacterized by the inability to resume UV inhibited DNA and RNAsynthesis. Two CS complementation groups (A and B) have been established. Athird group encompasses patients exhibiting both XP and CS symptoms, theybelong to XP groups B, D or G. The progressive neurological abnormalitiesassociated with CS may be due to the inability of CS cells to repairoxidatice DNA lesions (LePage et al., 2000).

PIBIDS is a photosensitive variant of Trichothiodystrophy (TTD) and the third syndrome that can be associated with NER defects(PIBIDS is the acronym of the characteristic clinical symptoms of thepatients for Photosensitivity, Ichthyosis, Brittle hair, Impairedintelligence, Decreased fertility and Short stature) (Itin et al., 2000).Certain mutations in the XPB and XPD genes have been shown to cause thePIBIDS phenotype, but not in combination with the specific XPcharacteristics like cancer proneness.

It has been shown that NER can operate via two subpathways. The firstpathway is global genome repair (GGR) and involves repair activity thatacts on DNA lesions across the genome. Although the efficiency of thispathway can be influenced by various parameters, it is not activelytargeted to specific regions of the genome. A second NER pathway is coupledto active transcription and is called transcription coupled repair. Thispathway involves repair activity that is directed to the transcribed strandof active genes.

The cloning of the XP genes and the isolation of the encoded proteins has lead to the elucidation of the core NER reactions and ultimately to the reconstitution of the process in vitro (Aboussekhra et al., 1995; Mu et al., 1995).

NER proteins and their functions

DNA damage recognition. Two proteins have been identified andimplicated in (one of) the first steps of NER, i.e. the recognition oflesions in the DNA: the XPA gene product and the XPC gene product incomplex with HR23B. In addition, the XPE protein has been shown to have ahigh affinity for damaged DNA, but whether it is required for the damagerecognition step of NER remains unclear. Cells from XPA patients areextremely sensitive to UV and have very low nucleotide excision repairactivity. In vitro the XPA protein binds preferentially to damaged DNAcompared to nondamaged DNA. The XPA protein binds to replication protein A(RPA) which enhances the affinity of XPA for damaged DNA and is essentialfor NER. The other complex that has been implicated in DNA damagerecognition is XPC-HR23B. XPC cells have low NER repair capacity, but theresidual repair has been shown to occur specifically in transcribed genes.It is very likely that the XPC-HR23B complex is the principal damagerecognition complex i.e. essential for the recognition of DNA lesions inthe genome (Sugasawa et al, 1998). Binding of XPC-HR23B to a DNA lesioncauses local unwinding, so that the XPA protein can bind and the wholerepair machinery can be loaded onto the damaged site. This would imply thatthe XPA protein has binding affinity for other repair proteins. Indeed, theXPA protein has been shown to bind to ERCC1 and TFIIH. The XPC-HR23Bcomplex is only required for global genome repair. In case of transcriptioncoupled repair when an RNA polymerase is stalled at a lesion, the DNA isunwound by the transcription complex and XPA can bind independently of XPC-HR23B complex.

XPE patients show mild dermatological symptoms and cells from thesepatients have a relatively high repair capacity. The function of the geneproduct is not completely clarified yet. Band shift assays suggested thatthe XPE gene product acts as a damaged DNA binding protein (DDB), with highaffinity to UV-induced 6-4PP. However, defective DDB binding activity isnot a common feature of XPE mutant cell lines and in fact two (or evenmore) proteins may be involved in the binding activity: p48 and p125. Incells from several XPE patient mutations in p48 have been found but so farno mutations have been found in the p125 gene. XPE cells are notnecessarily defective in repair: p125 is proposed to play a role in openingup chromatin to make CPD accessible to the NER machinery, but is notrequired for repair of 6-4PP. Interestingly, cell lines and primary tissuesfrom rodents are fully deficient in the expression of the p48 protein (Tanget al., 2000). This explains the absence of GGR of CPD in these cells.Exogenous expression of p48 in hamster cells confers enhanced removal ofCPD from genomic DNA and nontranscribed strand of active genes.

Damage demarcation. The striking discovery that subunits of basaltranscription factor TFIIH were involved in NER sheds light on a new aspectof NER : a close coupling to transcription via common use of essentialfactors. Two repair proteins, encoded by XPB and XPD genes, appeared to beidentical to components of the basal transcription factor TFIIH, a largecomplex involved in the initiation of transcription.The XPB and XPDproteins displayed 3'-5' and 5'-3' helicase activity respectively(Schaeffer et al., 1994). TFIIH fulfills a dual role in transcriptioninitiation and NER and the role of TFIIH in NER might closely mimic itsrole in the transcription initiation process. In transcription initiationTFIIH is thought to be involved in unwinding of the promoter site and toallow promoter clearance. In the NER process TFIIH causes unwinding of thedamage containing region that has been localized by XPC-HR23B and XPA-RPA,enabling the accumulation of NER proteins around the damaged site.

Among the XP patients, XPB patients are extremely rare (only 3patients known in the world) due to the fact that the XPB gene product isessential for transcription initiation and in all cases, these patientsshow the double symptoms of XP and CS. The helicase activity of XPD isindispensable for NER but not for transcription initiation. So , there ismuch more XPD patients, and only two patients have been described as XP andCS.

Incision. The XPF protein and the ERCC1 protein form a complex thatexhibits structure specific endonuclease activity that is responsible forthe 5' incision during the NER reaction. XPF-ERCC1 also binds to XPA(through ERCC1) and to RPA (through XPF) but not preferentially to damagedDNA. The XPG protein has DNA endonuclease activity without preference fordamaged DNA and is responsible for the 3' incision made during NER. At thesite of a lesion NER proteins create a DNA bubble structure over a lengthof approximately 25 nucleotides and the XPG protein incises the damaged DNAstrand 0-2 nucleotides 3' to the ssDNA-dsDNA junction. In most studies the3'-incision made by the XPG protein appeared to be made prior to andindependently of the 5'-incision by XPF-ERCC1. Patients belonging to theXP-G complementation group clinically exhibit heterogeneous symptoms, frommild to very severe, sometimes associated with CS. XP-G cells are almostcompletely repair-deficient and as UV-sensitive as XP-A cells. About halfof the described XPG patients exhibit also CS symptoms. In contrast to XPG,XP-F patients have a relatively mild XP phenotype without neurologicalabnormalities. Cells from XP-F patients are slightly UV-sensitive andexhibit low levels of repair initially after UV-irradiation.

Repair patch synthesis and ligation. Proliferating Cell NuclearAntigen (PCNA) is required for DNA synthesis by DNA polymerases delta andepsilon. PCNA has also been shown to be required for NER in vitro i.e. forthe DNA resynthesis step, suggesting that DNA polymerase delta or epsilonis involved in NER. Biochemical analysis and fluorescence microscopyrevealed that in quiescent cells upon UV-irradiation PCNA (that usuallyresides in the cytoplasm) becomes rapidly bound to chromatin. The enzymesinvolved in these pathways are normal in DNA repair-deficient cells.

Global genome repair (GGR)

GGR acts on DNA lesions throughout the genome, but the kinetics of repaircan be influenced by a number of parameters related to DNA lesion structureand chromatin configuration. It is conceivable that the damage recognitionstep is a rate-limiting step in the repair process and that more efficientrecognition of DNA lesions will lead to more rapid repair. The lesionrecognition and binding potency of proteins that are involved in damagerecognition, depends on the chemical structure of the DNA lesion itself orthe way it interferes with the DNA helical structure. Some lesions such asultraviolet light induced 6-4PP and CPD, are large bulky lesions located inthe minor groove of the DNA helix and are recognized by NER proteins asbeing abnormal structures in the DNA. DNA is thought to be a dynamicmolecule subject to an extremely rapid process of bending, twisting,unwinding and rewinding ('breathing'). Lesions that interfere with thesedynamic properties of the DNA may be recognized by repair proteins. Lesionsthat have been shown to be a good substrate for NER often cause localunwinding of a few DNA bases around the damaged site. UV-induced CPD aswell as cisplatinum-induced intrastrand crosslinks are a better substratefor in vitro NER when they are superimposed on a mismatch than in normallybase paired DNA. The unwinding of a few basepairs energetically favoursbending of the DNA and this may facilitate further unwinding by NERenzymes. Repair of DNA lesions that are substrates for NER by themselves,is strongly stimulated by disruption of base pairing at the site of thelesion.The role of chromatin structure in governing the repair efficiency isindicated by the notion that repair in the nontranscribed strand of activegenes or chromatin poissed for transcription, is faster than in inactive X-chromosomal genes (Venema et al., 1992). The latter are known to consist ofheavily methylated DNA sequences and their chromatin structure isrelatively inaccessible to molecular probes such as DNAse1. Thus, theefficiency of repair might be influenced by accessibility of DNA lesions torepair proteins. Indeed, when repair was investigated at the nucleotidelevel, profound differences in repair rate were found due to proteinbinding in promotor regions.

Transcription-coupled repair

The NER subpathway transcription-coupled repair (TCR) first describedby Mellon and Hanawalt for cultured mammalian cells (Mellon et al., 1987),specifically removes DNA lesions from the transcribed strand of an activegene. Subsequently, TCR was shown to operate in a variety of organismsincluding bacteria and yeast. All data indicate that TCR is directlycoupled to active transcription and it is generally assumed that a stalledtranscript provides a strong signal to attract the repair machinery. Allclassical XP cells are deficient in TCR except the group C that is fullydeficient in GGR but proficient in TCR (Van Hoffen et al., 1995).However,until now it is not clear how repair is coupled to transcription. A majorobstacle that prevents a major breakthrough, is the lack of a cell freesystem capable to perform TCR.

Genetic analysis has put some light on specific factors that play arole in TCR. In an E. coli mdf- mutant strain a protein has beenidentified called transcription-repair coupling factor (TRCF, the mdf geneproduct), that actively couples repair to a stalled RNA polymerase at thesite of a DNA lesion (Selby and Sancar, 1993). In mammalian cells suchfactor has not been found yet, but it was suggested that the proteinsmutated in the Cockayne' syndrome might fulfill such a function. Similarlyto the mdf bacteria strain, Cockayne syndrome cells are unable to performtranscription-coupled repair, whereas the global repair pathway isfunctioning normally. The defect in transcription-coupled repair has beenrelated to the inability of CS cells to restore UV-inhibited RNA synthesis(Mayne and Lehmann 1982). Slow removal of DNA lesions from transcriptiontemplates would prevent efficient transcription and this could lead to celldeath if essential genes are involved. Moreover, by analogy to bacteriasuch a factor could attract NER proteins. Indeed, several investigatorsshowed that CSB can be copurified with RNA polymerase II but could notdetect interaction of CSB with any other tested NER component. In cellsthat have been treated with UV, a small fraction of RNA polymerase IIbecomes ubiquitinated within 15 minutes after treatment and this fractionpersists for about 8 hours (Bregman et al., 1996). However, neither in CS-Anor in CS-B cells this specific response was observed. One explanationfavoured by several studies, is that the polymerase could be ubiquitinatedas a signal for degradation of the protein so that the lesion becomesaccessible for repair enzymes. In this model, CS proteins would be requiredto make lesions (at stalled transcripts) repairable.