Subpathways of nucleotide excision repair and their regulation - Nature

Oncogene (2002) 21, 8949 – 8956 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

Subpathways of nucleotide excision repair and their regulation Philip C Hanawalt*,1 1

Department of Biological Sciences, Stanford University, Stanford, California, CA 94305-5020, USA

Nucleotide excision repair provides an important cellular defense against a large variety of structurally unrelated DNA alterations. Most of these alterations, if unrepaired, may contribute to mutagenesis, oncogenesis, and developmental abnormalities, as well as cellular lethality. There are two subpathways of nucleotide excision repair; global genomic repair (GGR) and transcription coupled repair (TCR), that is selective for the transcribed DNA strand in expressed genes. Some of the proteins involved in the recognition of DNA damage (including RNA polymerase) are also responsive to natural variations in the secondary structural features of DNA. Gratuitous repair events in undamaged DNA might then contribute to genomic instability. However, damage recognition enzymes for GGR are normally maintained at very low levels unless the cells are genomically stressed. GGR is controlled through the SOS stress response in E. coli and through the activated p53 tumor suppressor in human cells. These inducible responses in human cells are important, as they have been shown to operate upon chemical carcinogen DNA damage at levels to which humans are environmentally exposed. Interestingly, most rodent tissues are deficient in the p53-dependent GGR pathway. Since rodents are used as surrogates for environmental cancer risk assessment, it is essential that we understand how they differ from humans with respect to DNA repair and oncogenic responses to environmental genotoxins. In the case of terminally differentiated mammalian cells, a new paradigm has appeared in which GGR is attenuated but both strands of expressed genes are repaired efficiently. Oncogene (2002) 21, 8949 – 8956. doi:10.1038/sj.onc. 1206096 Keywords: nucleotide excision-repair; transcriptioncoupled repair; global genomic repair; SOS response; p53; xeroderma pigmentosum; cockayne syndrome; UV sensitive syndrome; Li – Fraumeni syndrome Introduction Multiple strategies have evolved to minimize the genotoxic consequences of endogenous and environmental agents that damage DNA. The ubiquitous process of nucleotide excision repair (NER) removes a

*Correspondence: PC Hanawalt; E-mail: [email protected]

variety of structurally unrelated DNA lesions that are mutagenic and that may result in malignancy, faulty differentiation patterns and cell death. The efficiency of NER varies throughout the genome and different kinds of lesions are removed from the DNA with different efficiencies. Global genomic repair (GGR) is hindered or assisted by chromatin structure and proteins bound to the DNA. A dedicated subpathway, transcriptioncoupled repair (TCR), deals specifically with lesions which arrest the RNA polymerase in the transcribed strands of expressed genes. NER proceeds by a number of discrete enzymatic steps, as postulated four decades ago. These include recognition of a lesion in DNA; introduction of incisions in the damaged strand, one on each side of the lesion; removal of the oligonucleotide containing the lesion; resynthesis of the deleted nucleotide sequence using the complementary DNA strand as template; and finally, ligation of the newly-synthesized repair patch to the preexisting strand. A complex of proteins encoded by the uvrA, uvrB and uvrC genes is required for lesion recognition and incision in Escherichia coli. Helicase II (encoded by the uvrD gene), DNA polymerase I and ligase perform the subsequent steps. The complete series of reactions can be reconstituted in vitro from these six purified proteins (Sancar, 1996). Mammalian NER has also been reconstituted in vitro but this requires many more proteins to carry out essentially the same steps (reviewed in Lindahl and Wood, 1999). Even though the enzymatic details differ the principal features of NER are the same for organisms as diverse as bacteria, yeast and mammals. (Eisen and Hanawalt, 1999; Petit and Sancar, 1999; Zou and van Houten, 1999). The subpathways of NER The analysis of the repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in transcriptionally active versus inactive regions of the genome, and within transcribed and non-transcribed strands of expressed genes revealed the two distinct subpathways of nucleotide excision repair: TCR and GGR (Bohr et al., 1985; Mellon et al., 1987; Mellon and Hanawalt, 1989). TCR refers to the preferential repair of transcribed strands in active genes, and GGR refers to repair throughout the genome, including that in the non-transcribed strands of active genes. Repair rates in non-transcribed strands roughly reflect the overall

Excision repair regulation PC Hanawalt

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genomic average rates. For some lesions, like CPDs, TCR results in more rapid repair of the transcribed strands than the non-transcribed DNA strands, and this strand bias has usually been taken as the operational definition of TCR. However, for some other lesions, like the more structurally distorting 6-4 pyrimidine pyrimidone UV photoproducts (6-4PPs), which are very efficiently repaired by GGR, the effect of TCR may not be seen as a differential rate of repair in the two strands. Our initial studies revealed that while Chinese hamster ovary (CHO) cells are as fully proficient in TCR as human cells they are severely deficient in GGR of CPDs (Mellon et al., 1987). This was a confirmation of the so-called ‘rodent repairadox,’ that cultured rodent and human cells typically display similar clonal survival characteristics following UV irradiation, but that the cells from mice, rats, and hamsters are generally deficient in NER of CPDs (reviewed by Hanawalt, 2001). In E. coli the recognition of lesions in GGR is a dynamic two-stage process involving the UvrA and UvrB proteins (Zou et al., 2001). The initial recognition is of a distortion from the normal DNA helix. This is followed by a verification step in which the strand-opening activity of the UvrB helicase helps to determine that a nucleotide has been altered and establishes which strand has been damaged (reviewed in van Houten et al., 2002). In the case of TCR, the initiating event is the arrest of transcription at the site of the lesion (Mellon and Hanawalt, 1989). Then Mfd removes the blocked RNA polymerase from the DNA (thereby aborting the nascent transcript) and recruits UvrA to proceed with the same steps as with GGR (Selby and Sancar, 1993). In human cells, unlike bacteria, several of the proteins which operate in the recognition of lesions for GGR do not participate in TCR. These include XPC/HR23B and the UV DNA damage binding protein, UV-DDB (Tang and Chu, 2002; Hwang et al., 1999). For some lesions XPC/HR23B would appear to be the primary recognition protein (Sugasawa et al., 1998; Batty et al., 2000) while for others, like CPDs in particular, UV-DDB is additionally required and thought to initially enhance the DNA helix distortion so that XPC/HR23B can be more efficiently recruited (Tang and Chu, 2002; Wagasugi et al., 2002). XPA in association with the single strand binding complex RPA is recruited, as another damage-recognition complex that may participate in lesion verification, as it then recruits the TFIIH basal transcription initiation factor, which serves dual roles in transcription and in DNA repair. The component helicases of TFIIH, XPB and XPD, may be somewhat analogous to UvrB of E. coli in the role of lesion verification and strand determination, prior to the incision step. XPA is necessary for both GGR and TCR, but interestingly, it is not essential for a base-excision repair pathway for several oxidative DNA lesions that has been shown to include both GGR and TCR modes (Leadon and Cooper, 1993). There are a number of genes required for TCR of UV photoproducts that are not implicated Oncogene

in GGR. These include the Cockayne syndrome genes, CSA and CSB, and the gene responsible for ultravioletsensitive syndrome (Spivak et al., 2002). A growing number of genes have become implicated in the respective pathways of nucleotide-TCR and oxidative lesion-related TCR. Some genes are common to both of those pathways while others are specifically involved in one or the other. A current summary is provided by Spivak et al. (2002). It is important to appreciate, however, that the TCR pathway clearly does not operate upon some lesions such as 7-methyl guanine and 3-methyl adenine that are subject to efficient base excision repair (Plosky et al., 2002). Gratuitous NER in undamaged DNA? The broad substrate specificity of NER ranges from gross structural alterations in DNA to the minimal distortion caused by phosphorothiolate or methylphosphonate backbone modifications (Branum et al., 2001). Does excision repair ever attack undamaged DNA? In an early approach to answer this question we simply measured the background level of repair replication or ‘DNA turnover’ in E. coli which had not been exposed to any DNA damaging agent. Using the 5-bromouracil density-labeling protocol we detected a low level of repair replication (Couch and Hanawalt, 1967) and this was quantified in later studies at the level of roughly 0.02% of the nucleotides replaced per hour (Grivell et al., 1975). Curiously, we found that much of this ‘turnover’ was dependent upon transcription. That result was consistent with our documentation of rifampicin-sensitive ‘repair replication’ in E. coli following a period of thymine starvation (that is not known to introduce DNA lesions). We suggested that ‘transcription might sometimes involve the introduction of repairable single strand breaks in the bacterial DNA’ (Pauling and Hanawalt, 1965; Hanawalt et al., 1968). Following our discovery of TCR we have raised the possibility that the arrest of the RNA polymerase at a natural pause site in DNA might occasionally lead to gratuitous TCR. Such reiterative and futile repair replication might then cause significantly enhanced levels of ‘spontaneous’ mutagenesis in a frequently transcribed gene. The fidelity of repair synthesis may be compromised by the fact that the repair patches of only 12 – 13 nucleotides would not be subject to methyl-directed mismatch repair (Hanawalt, 1994). Although there is not yet any experimental evidence for gratuitous TCR, Sancar and his colleagues have recently shown that the Uvr-ABC system does indeed carry out a detectable level of excision from undamaged DNA in vitro, and furthermore that the excised fragments are 12 – 13 nucleotides long. In human cell extracts a similar activity on undamaged DNA yielded fragments of 23 – 28 nucleotides, again providing credibility that this represented the normal action of the NER system (Branum et al., 2001). It was concluded that DNA turnover due to these activities in vivo may contribute


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substantially to the genetic load of spontaneous mutagenesis. If this were a significant problem in vivo, an obvious way to minimize its impact would be to maintain the cellular concentrations of lesion recognition enzymes at very low levels until sensitive signaling mechanisms warned of a significant genotoxic threat. We have documented inducible GGR of CPDs in both E. coli and human cells, as consistent with this hypothesis (reviewed in Hanawalt et al., 2001). Inducible GGR in E. coli The arrest of replication forks at the sites of DNA lesions, leads to the induction of a large number of pleiotropic genes in E. coli through the SOS genomic stress response, that is controlled by RecA and LexA (Courcelle et al., 2001) (Table 1). Among the genes derepressed through this response in UV-irradiated E. coli are uvrA, uvrB, and uvrD. Interestingly, the gene normally responsible for dual incisions, uvrC, is not upregulated, but another incision gene is induced. That gene, called cho, encodes an incision nuclease that makes a 5’ side cut farther from the lesion than uvrC, thereby facilitating repair of larger lesions (Moolenaar et al., 2002). We have previously reported on the important role of the SOS response for the NER of CPDs (Crowley and Hanawalt, 1998). Elimination of the SOS response either genetically or by treatment with rifampicin (to inhibit transcription) strikingly reduced the efficiency of GGR of CPDs but had no

effect on the GGR of 6-4PPs. Mutants in which the SOS response was constitutively derepressed repaired CPDs more rapidly than did wild-type cells, and this rate was not affected by rifampicin. TCR of CPDs was seen in the absence of SOS induction but was undetectable when the SOS response was expressed constitutively, presumably because it was masked by very efficient GGR. These results suggested that damage-inducible synthesis of UvrA and UvrB is required for the efficient excision of CPDs and that the levels of these proteins determine the rate of NER of UV photoproducts. The upregulation of the uvrD helicase did not appear to be necessary for the enhanced efficiency of repair (Crowley and Hanawalt, 2001). Thus, the damage-inducible stress responses are generally critical for efficient GGR (but not TCR) of certain types of genomic damage (Crowley and Hanawalt, 1998). The results we obtained in SOS induced E. coli were remarkably parallel to those we had seen in human cells in response to p53 activation (Ford and Hanawalt, 1997). Inducible GGR in human cells Important insights into the control of lesion recognition in GGR were obtained through our analysis of the role of the p53 tumor suppressor in repair. It is wellknown that the accumulation and activation of p53 in response to DNA damage can lead to apoptosis or arrest of the cell cycle, presumably to provide time for

Table 1 Genes in E. coli with increased transcript levels following UV irradiation Repair, recombination and replication umuC, umuD (Pol V) dinB (Pol IV) polB (Pol II) uvrA, uvrB, uvrD (NER) cho (formerly ydjQ) (uvrC homologue) recA (homologous strand pairing; strand exchange) recN (DSB repair?) ruvA, ruvB (branch migration-Holliday jct) ruvC (resolvase-Holliday jct) recF (replication fork restart?) dnaA, dnaC, dnaN, rep (DNA replication genes)

206 76 36 36 36 106 206 26 1.46 26 26

Nucleotide metabolism nrdA, nrdB grxA upp

(ribonucleotide reductase subunit) (Glutaredoxin coenzyme) (uracil phosphoribosyltransferase)

26 56 26

Heat shock, transport ycgH, glvB potB, potC ipbB

(ATP-binding, transport) (spermidine, putrescine permease) (heat-shock protein)

66 26 26

Cell division sulA

(suppressor of lon; inhibits cell division)

106

Unknowns dinD, oraA, yebG, yigF, yigH, arpB

4 – 106

The average increase has been approximated from the data. All of the listed genes except those for nucleotide metabolism are dependent upon the SOS inducible response. (Adapted from Courcelle et al., 2001) Oncogene


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Figure 1 Recognition of photoproducts for NER in human cells. While both CPDs and 6-4PPs are readily recognized for TCR by RNAP II and 6-4PPs are efficiently targeted for GGR by XPC/hHR23, the less distorting CPDs require the additional factor UVDDB, prior to the actions of XPC/hHR23

repair of the damage before the cell divides or initiates a new round of replication. However, there are controversies regarding the role of p53 in apoptosis in different cellular systems and there are clearly apoptotic pathways which are independent of p53 (McKay and Ljungman, 1999; Dunkern and Kaina, 2002). Our work has established that p53 activation is required for efficient GGR of UV-induced CPDs in human fibroblasts (Ford and Hanawalt, 1995, 1997; Ford et al., 1998), as completely analogous to the effect of SOS induction in bacteria. Skin fibroblasts derived from tumors in patients with the cancer prone Li – Fraumeni syndrome, homozygous for mutations in the p53 gene, were remarkably defective in GGR of CPDs compared with that of related heterozygous mutants and normal cells (Ford and Hanawalt, 1995). When the expression of p53 in human fibroblasts was controlled, using a stably integrated tetracycline-regulated p53 gene, these observations were confirmed and it was established that the effect is specifically due to the p53 gene product (Ford and Hanawalt, 1997). Furthermore, we found that the GGR of CPDs is much more dependent upon p53 activity than is GGR of the more structure distorting 6-4 PPs. This suggested some heterogeneity in the requirement of p53 for GGR among different types of DNA lesions and that it would be worthwhile to explore this relationship for chemical carcinogens. Whereas UV irradiation is primarily associated with skin cancers, other genotoxins, such as the carcinogenic polycyclic aromatic hydrocarbons (PAHs), have numerous target organs. Humans are constantly exposed to PAHs as contaminants in the environment since they are formed during the inefficient combustion of fossil fuel. They are metabolized in human cells to electrophilic derivatives that form DNA adducts by interacting covalently with purine bases. These adducts Oncogene

are mutagenic and may well represent an early stage in PAH-induced carcinogenesis. Exposure to PAHs, due to smoking or other environmental factors that are associated with enhanced cancer risk, results in the formation of low levels of PAH-DNA adducts in various tissues (Beach and Gupta, 1992). We investigated the repair of adducts formed by benzo(a)pyrene7,8-diol-9,10-epoxide (BPDE), a reactive metabolite of the potent carcinogen benzo(a)pyrene that binds predominantly to the exocyclic amino position of guanine. Using the human fibroblasts in which expression of p53 could be regulated (noted above) and the ultrasensitive technique of 32P post-labeling we measured DNA adducts after exposure to BPDE levels as low as 0.1 mM. The BPDE-DNA adducts (at roughly 50 adducts/108 nucleotides) persisted for at least 3 days in cells deficient in p53 but were mostly repaired in cells expressing p53 (Lloyd and Hanawalt, 2000). It is important to appreciate that the levels of adducts measured were in the same range as those reported in biopsy tissue from smokers’ lungs. Strand-specific repair analyses confirmed our findings and additionally showed that TCR is unaffected in p53-deficient human cells exposed to BPDE (Wani et al., 2000). More recently we have extended our analysis to another widespread environmental contaminant and potent carcinogen, benzo(g)chrysene (B(g)CDE). Four major DNA adducts were detected, corresponding to the reaction of either the (+)- or (7)- anti-B(g)CDE stereoisomer with adenine or guanine. Exposure to a concentration of 0.01 mM resulted in a maximum of 20 adducts per 108 nucleotides in the p53 proficient cells at the 4 h time point (following removal of the agent) while 40 adducts per 108 nucleotide persisted at 24 h in the p53 deficient cells. All four adducts behaved similarly with respect to the effect of p53 expression upon their removal. Thus, p53 appears to minimize the


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appearance of B(g)CDE adducts in human cells by upregulating global NER and thereby reducing the maximum adduct levels attained. These results for several important chemical carcinogens are very similar to those that we had reported earlier for CPDs (Ford and Hanawalt, 1997) and they have established the generality of the p53 role in GGR. What is the mechanism of the p53 effect on GGR efficiency? In vitro studies have revealed no direct effect of p53 upon NER. The p53 effect on GGR of CPDs is mediated in large part through p48, a protein involved in DNA damage recognition that is missing in most XPE cell lines and in CHO cells (Hwang et al., 1999; Tang and Chu, 2002). The p48 protein is a component of UV-DDB and we have shown that expression of the p48 gene is upregulated in UV-irradiated human cells in a p53 dependent manner (Hwang et al., 1999). Thus, it is a transactivating role of p53 that is implicated in the control of efficient GGR. In more recent studies it was shown that transfection of the human p48 gene into CHO cells confers UV-DDB and enhances the removal of CPDs from the genomic DNA by GGR (Tang et al., 2000). Therefore, p48 appears to be a link between p53 and efficient GGR in mammalian cells. However, p53 is also involved in transactivation of other genes implicated in the early steps of NER, such as gadd45. Loss of gadd45 also results in deficient GGR of CPDs, and there is some evidence that gadd45 is involved in chromatin remodeling concurrent with DNA repair (Smith et al., 2000). Adimoolam and Ford (2002) have recently shown that the mRNA and protein product of the XPC gene are UV inducible in human WI38 fibroblasts and in HCT116 colorectal cancer cells with normal p53 expression. In contrast, no significant upregulation of XPC was detected in p53 deficient derivatives of those cells. Thus, as again parallel to the results with E. coli, it is the proteins implicated in lesion recognition for GGR that are inducible by genomic stress. An interesting and potentially significant technical point is that essentially all of the UV studies reported above have used nearly monochromatic 254 nm light. Drobetsky and colleagues have reported that following UVB (290 – 320 nm) exposure p53-deficient human lymphoblastoid cells are deficient in TCR as well as GGR (Therrien et al., 1999). The broad band of UVB wavelengths is more environmentally relevant than is monochromatic 254 nm. UVB also produces a different spectrum of UV photoproducts and interacts significantly with other molecules in addition to nucleic acids. In apparent contrast to the results for human cells and for E. coli a recent systematic analysis in Saccharomyces cerevisiae (that contains no p53 homologue!) led to the conclusion that few, if any, of the genes involved in repairing DNA lesions are induced in response to cellular exposure to the agents that produce them (Birrell et al., 2002). In particular there was no evidence that genes for lesion recognition in

NER were upregulated. Thus, yeast may be able to accommodate the likely deleterious effects of gratuitous repair inflicted by the constitutive levels of lesion recognition enzymes. Some tumor viruses interfere with inducible GGR Tumor virus infection of some types can result in the abrogation of p53 and can correspondingly reduce the efficiency of GGR. When p53-deficiency was conferred in human primary fibroblasts by enhancing p53 degradation, through expression of the papillomavirus E6 gene, we observed a major reduction in the GGR of CPDs and a lessor reduction in the removal of 64PPs (Ford et al., 1998). In SV40-transformed human fibroblasts, in which the large T-antigen interferes with p53 function, we also observed a very significant reduction in the GGR of CPDs. That deficiency was documented by three different assays and in several different SV40 transformed cell lines, and this result was in contrast to the proficient GGR expressed in the non-transformed parental cells. There was no significant effect of p53 deficiency upon TCR in any of these cases (Bowman et al., 2000). A third example is provided by the hepatitis Bx virus which also interferes with p53 function. Unlike cells from most rodent tissues the hepatocytes in mice exhibit proficient GGR of CPDs. Thus, it is likely that p53 transactivation of p48 and inducible UV-DDB activity is normal. Yet, repair of CPDs was strikingly diminished in hepatocytes in which the hepatitis Bx gene was expressed (Prost et al., 1998). The induced genomic instability and tumorogenic effect of the viruses in each of the cited examples could be a consequence of compromised GGR in the infected cells. Most rodent tissues are deficient in p53 regulated NER Shortly after the discovery of repair replication as the patching step in NER (Pettijohn and Hanawalt, 1964), it became apparent that cells from different species respond to the same dose of UV with vastly different rates and extents of repair replication (Painter and Cleaver, 1969; Trosko et al., 1965). At that time it was assumed that the amount of repair replication after a given UV dose must reflect the ability of the cells to survive the damage, and that the efficiency of CPD repair was indicative of the overall cellular response to many different types of damage. However, excision of CPDs was not detected at all in UV irradiated mouse L cells (Klimek, 1965). Similar results were reported for Chinese hamster cells (Trosko et al., 1965; Lohman et al., 1976). We reported that while human HT1080 fibroblasts typically removed 50 – 80% of the T4 endonuclease V sensitive sites (used as a quantitative indication of CPDs), mouse PG19 fibroblasts removed only 10 – 30%, within 24 h after 5 J/m2 UV. However, the colony-forming ability of the UV-irradiated mouse Oncogene


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cells was very similar to that of the human cells over the range of 1 – 10 J/m2. We concluded that ‘mouse cells can survive a higher level of unrepaired damage than can human cells’ (Ganesan et al., 1983). Chinese hamster V79 fibroblasts in culture were also shown to be severely deficient in removal of CPDs from the genome (van Zeeland et al., 1981), as were cultured rat fibroblasts (Vijg et al., 1984). Of course the repair of 64 PPs was not taken into account and the TCR pathway had not yet been discovered when these studies were conducted. One can ask to what extent the results obtained for CPD repair in cultured cells might reflect those from the intact tissue in vivo? In a comparison of CPD repair in human epidermis with confluent cultures of keratinocytes from the same subjects, we reported similar repair kinetics using an immunoassay to measure the amount of DNA in each fraction of alkaline sucrose gradients, to determine the size distribution of DNA molecules after treatment with the T4 endonuclease V (Hanawalt et al., 1987). Another immunological approach to detect DNA in fractions from alkaline sucrose gradients was utilized by Mullaart et al. (1988) who found that cultured rat epidermal keratinocytes removed only 20% of the CPDs in 24 h while 50 – 60% of the CPDs were removed from the intact epidermis within 3 h. It was concluded that ‘the capacity of rat skin cells to remove pyrimidine dimers is almost completely lost upon transfer of these cells into culture.’ In summary, it would appear that, at least in some situations, the efficiency of CPD repair may be attenuated as epidermal cells are explanted from rodent skin. Therefore, one should be cautious about extrapolating results from DNA repair efficiencies measured in cultured cells to the case in vivo. Early subcultures of mouse embryo fibroblasts were shown to remove CPDs at rates similar to (or somewhat lower than) those observed in human cells, but excision of CPDs was reported to decline abruptly after the fourth to the sixth subculture and, in the permanent 3T3 mouse cell line, there was no detectable excision (Ben-Ishai and Peleg, 1975; Peleg et al., 1976). It was suggested that the ‘cessation of excision repair may be due to genetic repression.’ It is likely that this hypothesis is correct, based upon more the recent studies cited above (cf. Hwang et al., 1998; Tang et al., 2000; Tang and Chu, 2002). That, however, does not resolve the paradoxical lack of correlation between clonal survival and repair. Cell survival would appear to be more generally dependent upon TCR than upon GGR for some types of lesions. This may be a consequence of the fact that an arrested RNA polymerase II at a lesion constitutes a strong signal for the apoptotic response as well as for p53 activation. Thus, cells deficient in TCR are much more sensitive to UV-induced apoptosis than are TCR proficient cells (Ljungman and Zhang, 1996). Then, the low skin cancer susceptibility in Cockayne syndrome (CS) (characterized by a deficiency in TCR but not GGR) could be Oncogene

a consequence of apoptosis, since dead cells don’t form tumors. That hypothesis is supported by the comparison with xeroderma pigmentosum (XP) group C, in which the cells are proficient in TCR but deficient in GGR. In XPC the signal for p53 activation and apoptosis is much weaker than that in CS but the GGR deficiency would be expected to result in high mutation rates and oncogenic transformation in the surviving cells (cf. Dumaz et al., 1997). In contrast, the surviving cells in the case of CS are fully proficient in GGR which will eventually deal with most of the lesions, thereby reducing mutation rates and precluding oncogenesis. DNA repair in terminally differentiated neurons: a new paradigm The accumulation of deleterious alterations in neuronal DNA has often been invoked in models for neurological diseases and aging. However, there have been relatively few definitive studies to test these models. We have studied NER of UV induced DNA damage in terminally differentiated human neurons for comparison to that in their precursor cells. A striking attenuation of GGR was noted in adult differentiated neurons (Nouspikel and Hanawalt, 2000). In human fetal neurons GGR was initially normal but then, as cultures matured, a similar, although less complete, impairment in GGR was observed (Nouspikel and Hanawalt, 2002). One could suppose that neurons do not repair most of their DNA because they do not need to replicate or transcribe most of their genome. Nevertheless, they must maintain the integrity of those genes that are being expressed, and in fact, we found transcribed genes very efficiently repaired in the differentiated neurons. Unlike the situation for TCR in growing cells, in which the transcribed strand is preferentially repaired, we have shown that both DNA strands of active genes are repaired efficiently in these nondividing cells (Nouspikel and Hanawalt, 2000). We have postulated the existence of a dedicated repair mechanism, which we have termed ‘differentiation associated repair’ (DAR), that efficiently repairs the non-transcribed strand of active genes in terminally differentiated cells. DAR may be required in cells in which GGR has been attenuated, because the nontranscribed strand serves as the template to repair the transcribed strand. If lesions were to accumulate in the non-transcribed strand of active genes over an extended period, it would become increasingly likely that TCR would fail when using that strand as template for repair. We suggest that DAR prevents such a situation, which would otherwise result in agerelated inactivation of many genes in terminally differentiated cells, causing progressive metabolic dysfunction. Failures in the proposed DAR pathway are likely to result in premature neuron ‘aging’ and apoptosis, leading to early dementia (Nouspikel and Hanawalt, 2002, 2003).


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Our analysis of gene expression profiles by quantitative RT – PCR has revealed that the genes encoding the structure-specific nucleases (XPG and XPF/ ERCC1) needed for the dual incisions in NER are remarkably upregulated in adult neurons but there is no significant downregulation of XPC, that might have been consistent with attenuated GGR (Nouspikel and Hanawalt, 2000). It will be of interest to determine the roles of XPC and UV-DDB in the proficient repair of expressed genes in neurons.

Acknowledgements The research in my laboratory has been supported by an Outstanding Investigator Grant (CA 44349) and more recently by grants (CA77712, CA91456 and CA90915) from the National Cancer Institute. I am indebted to my students and colleagues, many of whom are cited in the references, for their remarkable contributions to the ideas as well as the experimental results presented in this review. I wish to thank Denise Flowers for her essential help with the manuscript preparation.

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