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tumor development was absent in a group of 22 control animals [26]. The high frequency of hepatocellular adenomas indicates that the liver accumulates high levels of NER lesions or is particularly sensitive to them, a notion that will be of importance in the discussion of the phenotype of ERCCl -deficient mice later. Oral administration of the carcinogen B[a]P to XPA mice induced mainly lymphomas with reduced latency and higher frequency than wild-type mice, establishing XP as a disease with increased risk to internal tumors. To study the correlation between carcinogenesis and mutagenesis, XPA mice were intercrossed with transgenic mice carrying multiple copies of a plasmid containing the lacZ marker gene in their genome. After oral administration of B[a]P, plasmids were rescued from the genome and mutation frequencies in the lacZ gene were detelmined, revealing a con'Clation between lymphoma development and increased mutation fi:equency in lymphogenic tissue of XPA mice [26]. Similarly, mutagenesis at the endogenous hprtlocus in B[a]P-h'eated XPA mice correlated with Iymfomagenesis [27]. Because XPA mice are sensitive to a broad range of genotoxic agents (including UV, B[a]P, PhIP, N-OH-AAF, mitomycin C, cis-platin), they are particularly suited for screening genotoxic agents. Therefore, this mouse mutant is now evaluated in a program of the phannaceutical industry and US government with various carcinogenic and genotoxic compounds, honnones and inununo-suppressing carcinogens. Together with p53+1- mice, transgenic II-ras mice, and several other models, XPA mice are considered as an alternative model for testing carcinogenic and inununo-suppressive compounds. The reader is refened for more information about the test program to web-site Http://ntp-server.lliehs.nih.gov/Main---'pages/ transgen/lLSItbl2.hhnl. CONSEQUENCES OF MULTI-FUNCTIONALITY OF NER PROTEINS Besides their role in nucleotide excision repair, many individual NER proteins are simultaneously also involved in other cellular processes. As mentioned before, the TCR subpathway accomplishes preferential repair of transcription-blocking lesions from transcribed DNA sequences, involving the CSA and CSB proteins. Recently, direct interaction between the CSB protein and the (elongating) RNApolII complex has been observed [28]. This implies that a CSB defect may affect the process of transcription elongation as welL The coupling between NER and transcription appeared even more intimate when it was discovered that the XPB and XPD genes encode the helicase subunits of the dual functional DNA repair/basal transcription factor TFIIH [29, 30] implying that XPB and XPD are also basal transcription initiation proteins. Other NER components are involved in mitotic recombination and replication or have likely connections with base excision repair, ubiquitination and cell cycle regulation (see Table II for an overview). Predictably, the close link of NER with so many different processes will have multiple effects on the physiology of an organism when any of these factors is defective. In the next 36 Chapter 2
section, the phenotype of several of the NER-deficient mice is described, paying special attention to the role of endogenous DNA damage in the onset of some of the pathologic symptoms. ERCCl, premature aging and the stress response The ERCCI gene was the first human NER gene to be isolated via an NERdeficient Chinese hamster cell mutant as recipient for transfection cloning [31]. In contTast to other human NER genes cloned following the same strategy, subsequent studies excluded that ERCCI is involved in any known complementation group of the three human NER syndromes. This suggests that mutations in this gene are either very rare, lethal or induce an unexpected phenotype. The EReC I protein complexes with the XPF product and the resulting heterodimer has a stmcturespecific endonuclease activity incising the 3'-extending single strand at a double strand to single strand transition in DNA [32]. In the NER reaction mechanism this corresponds with the 5' incision of the damaged strand. In addition to NER, the ERCC lIXPF complex has a function in a mitotic recombination process that presumably is responsible for repair of intrastrand crosslinks. Chinese hamster ERCCI and ERCC4 (XPF) mutants are uniquely hypersensitive to DNA crosslinking agents such as mitomycin C (MMC) [33] and ERCCI-deficient mouse embryonic stern (ES) cells cany a defect in gene targeting by homologous recombination when the targeting constmct harbors heterologous ends (G. Weed a, unpublished observation). Similarly, MEFs isolated from ERCCI knockout mice Table II. Presumed or established involvement of NER proteins in diverse cellular processes NER compollell! TFI!I-I, CSB, CSA eSB, CSA, XPG (TfllH) [{]lA, PCNA, DNA poJymerases, ligase I RAD23a!b ERCCIIXPF Process Basal Transcription, Cell Cycle Regulation l3ase Excision Repair Replication Ubiquitination Cross Link Repair! Mitotic Recombination displayed besides a complete NER defect also sensitivity to crosslinking agents and an enhanced spontaneous and induced mutation rate [34]. Weeda et al reported that ERCCI MEFs display premature cellular senescence, with large polyploid nuclei in early passages, and many non-cycling cells [8]. Interestingly, neither established ERCCI-deficient hamster cells, nor ERCCI-/- ES cells (Weeda, unpublished data) display this phenotype, suggesting a relation between mortallimrnortal status of the cell. Premature cellular senescence is also not observed in any of the MEFs of the other NER mouse models analyzed (J. de Wit, unpublished data). Life span of ERCCI homozygote mouse mutants is strongly reduced. Two independent studies NER-deficient mouse models 37
Page 1 and 2: A MOUSE MODEL FOR TRICHOTHIODYSTROP
Page 3 and 4: CONTENTS Aim of the thesis 5 Chapte
Page 5: AIM OF THE THESIS Nucleotide excisi
Page 9 and 10: NUCLEOTIDE EXCISION REPAIR AND HUMA
Page 11 and 12: capacity to generate bulky base add
Page 14 and 15: strong interaction between XPG and
Page 16 and 17: XP is characterized by genetic hete
Page 18: are diagnosed as a collodion baby (
Page 22 and 23: 23. Gerard, M., Fischer, L., Moncol
Page 24 and 25: associatcd with mutations in the DN
Page 27: CHAPTER 2 MOUSE MODELS TO STUDY THE
Page 30: NER DEFICIENCY AND GENOTOXIC SENSIT
Page 34 and 35: XPC-I-p53+1- mice were more aggress
Page 38 and 39: [8, 9] reported substantial embryon
Page 41 and 42: in male sterility. As suggested by
Page 43: A point of concern in the extrapola
Page 47: CHAPTER 3 DISRUPTION OF THE MOUSE X
Page 50: INTRODUCTION To counteract the dele
Page 57: RAD3 and RAD25 protein (the latter
Page 63: CHAPTER 4 A MOUSE MODEL FOR THE BAS
Page 72 and 73: Death occurs after a short period o
Page 77: Table 3. Comparison of the TTDIBEL
Page 81 and 82: model (UDS, UV survival, pathology,
Page 84 and 85: 33. Stefanini, M., Giliani, S., Nar
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INTRODUCTION Genomic instability is
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correlates very well with literatur
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mice, which are histologically noml
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proneness in mice cOlTesponds with
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18, Lehmann, A. R., C. F, Arlclt, B
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CHAPTER 6 SYMPTOMS OF PREMATURE AGI
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INTRODUCTION The genome is under co
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osteosclerosis, carly depigmentatio
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oocyte that showed gemlinal vesicle
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DISCUSSION Human aging syndromes Ag
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lower levels of the anti-oxidants s
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6. Nance, M.A and Ben·y, S.A. (199
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SUMMARY AND CONCLUSIONS DNA damage
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features are due to an impairment o
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symptoms in normal aging. Oxidative
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In Hoofdstuk 5 worden experimenten
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LIST OF PUBLICATIONS Bitter, W., va
Page 134 and 135:
DANKWOORD Het schrijven van een dan
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