Alain Sarasin

The ERCC2/DNA repair protein is associated with the class II BTF2/TFIIH transcription factor.

ERCC2 is involved in the DNA repair syndrome xeroderma pigmentosum (XP) group D and was found to copurify with the RNA polymerase II (B) transcription factor BTF2/TFIIH that possesses a bidirectional helicase activity. Antibodies directed towards the 89 kDa (ERCC3) or the p62 subunit of BTF2 are able to either immunoprecipitate ERCC2 or shift the polypeptide in a glycerol gradient. Conversely, an antibody directed towards ERCC2 also retains or shifts BTF2. ERCC2 could be resolved from the other characterized components of BTF2 upon salt treatment, while its readdition enhanced BTF2 transcription activity. ERCC2, ERCC3 and p44 are three repair proteins found in association with BTF2. Two of them, ERCC2 and ERCC3, are responsible for atypical forms of XP disorders which confer a high predisposition to skin cancer. This includes clinical features that lack an adequate rationalization on the basis of nucleotide excision repair (NER) deficiency but which may now be explained better in terms of a partial transcription deficiency.

RETRACTED: Transcription-Coupled Repair of 8-oxoGuanine

Analysis of transcription-coupled repair (TCR) of oxidative lesions here reveals strand-specific removal of 8-oxo-guanine (8-oxoG) and thymine glycol both in normal human cells and xeroderma pigmentosum (XP) cells defective in nucleotide excision repair. In contrast, Cockayne syndrome (CS) cells including CS-B, XP-B/CS, XP-D/CS, and XP-G/CS not only lack TCR but cannot remove 8-oxoG in a transcribed sequence, despite its proficient repair when not transcribed. The XP-G/CS defect uniquely slows lesion removal in nontranscribed sequences. Defective TCR leads to a mutation frequency at 8-oxoG of 30%-40% compared to the normal 1%-4%. Surprisingly, unrepaired 8-oxoG blocks transcription by RNA polymerase II. These data imply that TCR is required for polymerase release to allow repair and that CS results from defects in TCR of oxidative lesions.

UV irradiation triggers ubiquitin-dependent degradation of p21WAF1 to promote DNA repair

p53-mediated increase in cyclin-dependent kinase inhibitor p21(WAF1) protein is thought to be the major mediator of cell cycle arrest after DNA damage. Previously p21 protein levels have been reported to increase or to decrease after UV irradiation. We show that p21 protein is degraded after irradiation of a variety of cell types with low but not high doses of UV. Cell cycle arrest occurs despite p21 degradation via Tyr(15) inhibitory phosphorylation of cdk2 and differs from the classical p21-dependent checkpoint elicited by ionizing radiation. In contrast to the basal turnover of p21, degradation of p21 switches to ubiquitin/Skp2-dependent proteasome pathway following UV irradiation. ATR activation after UV irradiation is essential for signaling p21 degradation. Finally, UV-induced p21 degradation is essential for optimal DNA repair. These results provide novel insight into regulation of p21 protein and its role in the cellular response to DNA damage.

ERCC1 codon 118 polymorphism is a predictive factor for the tumor response to oxaliplatin/5-fluorouracil combination chemotherapy in patients with advanced colorectal cancer.

Purpose: The aim of our study was to assess whether the polymorphism of the nucleotide excision repair enzyme, excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1), had an effect on the tumor response in patients treated with standard chemotherapy regimens for a metastatic colorectal cancer. We have studied the synonymous polymorphism that causes a single nucleotide change C to T at codon 118 converting a codon of common usage (AAC) to a less used codon (AAT), both coding asparagine. This change results in a decreased ERCC1 gene expression, which impairs repair activity. Experimental Design: Ninety-one patients with a median age of 55.1 years treated for a metastatic colorectal cancer were included in our retrospective study. The ERCC1 polymorphism was analyzed in the normal tissue of all patients. Results: Twenty (22%) were homozygous for AAC codon (C/C genotype), 30 were (33%) homozygous for AAT codon (T/T genotype), and 41 (45%) were heterozygous (C/T genotype). The objective response rate to oxaliplatin in combination with 5-fluorouracil (5-FU) was significantly higher in the T/T genotype group compared with the C/T and the C/C genotype groups (61.9%, 42.3%, and 21.4%, respectively; P = 0.018). By contrast, no significant difference was observed when patients were treated with either 5-FU alone (45%, 29.2%, and 33.3%, respectively; P = 0.407) or in combination with irinotecan (46.1%, 25.0%, and 27.3%, respectively; P = 0.305). Conclusions: Our observations allowed us to define the first useful predictive criterion for oxaliplatin/5-FU response in patients with metastatic colorectal cancer.

The molecular pathways of ultraviolet-induced carcinogenesis

Cancer development requires the accumulation of numerous genetic changes which are usually believed to occur through the presence of unrepaired DNA lesions. Exogenous or endogenous DNA-damaging agents can lead to mutations in the absence of efficient error-free repair, via replication of DNA damage. Several DNA repair pathways are present in living cells and well-conserved from bacteria to human cells. The nucleotide excision repair (NER), the most versatile of these DNA repair systems, recognizes and eliminates a wide variety of DNA lesions and particularly those induced by ultraviolet (UV) light. The phenotypic consequences of a NER defect in humans are apparent in rare but dramatic diseases characterized by hypersensitivity to UV and a striking clinical and genetic heterogeneity. The xeroderma pigmentosum (XP) syndrome is a human disorder inherited as an autosomal recessive trait. Persistence of unrepaired DNA damage produced by exposure to UV light is associated, in the XP syndrome, with an extremely high level of skin tumors in sun-exposed sites. Several key genes are mutagenized by UV-light and are responsible for skin cancer development. Mutations are found on ras oncogenes, p53 and PTCH tumour suppressor genes in skin cancers from DNA repair proficient as well as XP patients. The typical signature of UV-induced mutations found on these genes allows one to conclude that the uvB part of sunlight is responsible for the initiation of the carcinogenesis process.

XPD Mutations Prevent TFIIH-Dependent Transactivation by Nuclear Receptors and Phosphorylation of RARα

Inherited mutations in the XPD subunit of the general transcription/repair factor TFIIH yield the rare genetic disorder Xeroderma pigmentosum (XP), the phenotypes of which cannot be explained solely on the basis of a DNA repair defect. In cells derived from XP-D patients, we observed a reduction of the ligand-dependent transactivation mediated by several nuclear receptors (RARalpha, ERalpha, and AR). We demonstrate that the XPD mutation alters cdk7 function in RARalpha phosphorylation. Transactivation is restored upon overexpression of either the wild-type XPD or the RARalphaS77E (a mutation which mimics phosphorylated RARalpha). Thus, we demonstrate that the cdk7 kinase of TFIIH phosphorylates the nuclear receptor, then allowing ligand-dependent control of the activation of the hormone-responsive genes.

High expression of DNA repair pathways is associated with metastasis in melanoma patients.

We have identified a gene-profile signature for human primary malignant melanoma associated with metastasis to distant sites and poor prognosis. We analyse the differential gene expression by looking at whole biological pathways rather than individual genes. Among the most significant pathways associated with progression to metastasis, we found the DNA replication (P=10−14) and the DNA repair pathways (P=10−16). We concentrated our analysis on DNA repair and found that 48 genes of this category, among a list of 234 genes, are associated with metastatic progression. These genes belong essentially to the pathways allowing recovery of stalled replication forks due to spontaneous blockage or induced DNA lesions. Because almost all these differentially expressed repair genes were overexpressed in primary tumors with bad prognosis, we speculate that primary melanoma cells that will metastasize try to replicate in a fast and error-free mode. In contrast to the progression from melanocytes to primary melanoma, genetic stability appears to be necessary for a melanoma cell to give rise to distant metastasis. This overexpression of repair genes explains nicely the extraordinary resistance of metastatic melanoma to chemo- and radio-therapy. Our results may open a new avenue for the discovery of drugs active on human metastatic melanoma.

The specificity of p53 mutation spectra in sunlight induced human cancers.

Ultraviolet (UV) irradiation emitted by the sun has been clearly implicated as a major carcinogen in the formation of skin cancers in man. Indeed, the high levels of cutaneous tumors in xeroderma pigmentosum patients (XP) who are deficient in repair of UV-induced lesions have confirmed that DNA damage produced by sunlight is directly involved in the cancer development. The tumor suppressor gene, p53, very frequently found modified in human cancers, has proved to be a perfect target gene for correlating mutation spectra with different cancer causing agents as there are nearly 300 potential mutation sites available for analysis. In a comparative analysis of p53 mutations found in internal cancers with those in skin tumours we show here that clear differences exist between the types of spectra obtained. The specificity of UV induced mutations in skin cancers is confirmed when single and tandem mutations are compared. Most of the p53 point mutations found are GC to AT transitions both in skin and internal tumors where in the latter they are located mainly at CpG sequences probably due to the deamination of the unstable 5-MeC. Moreover, mutations are targeted at py-py sequences in over 90% of skin tumors whereas in internal cancers the distribution is proportional to the frequency of bipyrimidine sequences in the p53 gene. Most significantly, all mutations found in XP skin tumors are targeted at py-py sites and more than 50% are tandem CC to TT transitions considered as veritable signatures of UV-induced lesions. Tandem mutations are also relatively common (14%) in skin tumors from normal individuals compared to their very rare occurrence in internal malignancies (0.8%). Finally, nearly all mutations observed in XP skin tumors are due to unrepaired lesions remaining on the coding strand whereas no strand bias is seen in mutation location of internal or skin tumors from normal individuals. In fact the mutation spectrum analysed in XP skin cancers has permitted the first demonstration of the existence of preferential repair in man. In conclusion, using the p53 gene as a probe it is obvious that the mutation spectra from skin tumors are very similar to those observed in UV-treated gene targets in model systems but statistically different from those described in other types of human cancer. This has allowed us to demonstrate, without ambiguity, the major role of UV-induced DNA lesions in sunlight related skin carcinogenesis.

Variability in nucleotide excision repair and cancer risk: a review

Cancer initiation is classically associated with the induction of mutations on specific oncogenes or tumor suppressor genes, due to the presence of unrepaired DNA lesions produced by endogenous or exogenous genotoxic agents. Among several DNA repair pathways, the nucleotide excision repair (NER) is the most important and versatile one in removing the bulky adducts induced by physical and chemical carcinogens. Xeroderma pigmentosum (XP), characterized by a deficiency in NER and an over 1000-fold increased risk of skin cancer, represents a paradigm to understand the role of unrepaired lesion in the development of cancer. We reviewed here several NER assays used in epidemiological studies investigating the association between DNA repair efficiency and cancer risk. Reduced DNA repair could contribute to the development of cutaneous basal cell carcinoma (BCC), although discordant results have been reported. More consistent findings were observed between cellular sensitivity towards genotoxic agents and smoking-related cancers.

Molecular analysis of mutations in DNA polymerase η in xeroderma pigmentosum-variant patients

Xeroderma pigmentosum variant (XP-V) cells are deficient in their ability to synthesize intact daughter DNA strands after UV irradiation. This deficiency results from mutations in the gene encoding DNA polymerase η, which is required for effecting translesion synthesis (TLS) past UV photoproducts. We have developed a simple cellular procedure to identify XP-V cell strains, and have subsequently analyzed the mutations in 21 patients with XP-V. The 16 mutations that we have identified fall into three categories. Many of them result in severe truncations of the protein and are effectively null alleles. However, we have also identified five missense mutations located in the conserved catalytic domain of the protein. Extracts of cells falling into these two categories are defective in the ability to carry out TLS past sites of DNA damage. Three mutations cause truncations at the C terminus such that the catalytic domains are intact, and extracts from these cells are able to carry out TLS. From our previous work, however, we anticipate that protein in these cells will not be localized in the nucleus nor will it be relocalized into replication foci during DNA replication. The spectrum of both missense and truncating mutations is markedly skewed toward the N-terminal half of the protein. Two of the missense mutations are predicted to affect the interaction with DNA, the others are likely to disrupt the three-dimensional structure of the protein. There is a wide variability in clinical features among patients, which is not obviously related to the site or type of mutation.