DNA repair, DNA replication and human disorders: A personal journey

I was born in 1946 and grew up in the industrial north-west of England close to the city of Manchester. My parents were German- Jewish refugees, who left Germany fairly early, in 1933. My father helped to establish and was one of the directors of a tannery, which made leather for shoes and handbags. This was part of a group of tanneries established first in Strasbourg by my great-grandfather Ferdinand Oppenheimer. I would describe my childhood and adolescent years as comfortable by general post-war standards. I went to a state primary school and obtained a scholarship to Manchester Grammar School (MGS), a fairly prestigious secondary school. As a child I was always interested in chemistry but had little interest in or knowledge of biology. The educational system in the UK at that time was such that one had to specialise very early and as a consequence I have had no formal biology education since the age of 12, something I have managed to hide reasonably successfully for the rest of my life! In my final two years at MGS I studied just physics, chemistry and mathematics and obtained a scholarship to Pembroke College, Cambridge (England) to study Natural Sciences, with the intention of becoming a chemist. In the second year at Cambridge, one of the options was a course on biochemistry. Having no real idea what this was, I read a book about it in the summer of 1965, and was truly astonished and excited to discover that the basis of life was just a bunch of rather complicated organic chemistry reactions. So I took the biochemistry course in my second year. By the end of that year, I was fed up with chemistry and for my final year I chose to do biochemistry rather than chemistry, a decision I have not regretted. The biochemistry lectures must have been pretty up-to-date, as we were told briefly about the discovery of DNA repair by Dick Setlow [1], a topic that seemed rather esoteric at the time

Characterization of the multifunctional XPG protein during Nucleotide-excision-repair

Xeroderma pigmentosum (XP), a cancer model disease, is the perfect proof for the existing model of carcinogenesis activated by mutations. All patients share a defect in Nucleotide excision repair (NER). The gene, which is disease-causing for XP complementation group G (XPG) patients, encodes for the multifunctional endonuclease XPG. This enzyme has many binding partners like TFIIH, RPA and PCNA, and acts at a crucial step at the very end of NER. Several functional domains of XPG were mutated to investigate the behavior of the respective mutants during NER intermediates of dual incision, using DNA repair synthesis (UDS) and Host cell reactivation (HCR) assays. Furthermore, a new XPG patient with implications for the functional XPG-TFIIH interaction has been studied. By genotype-phenotype correlation of a XPG patient (XP172MA), this study greatly suggests to narrow down the functionally important XPG interaction domain between TFIIH and XPG to the XPG amino-acids 30-85. This study demonstrates that the functional PCNA-XPG interaction is more important for NER than the endonuclease function of XPG. The C-terminally located PIP-box of XPG is required for immediate UV response but not for the functionality of XPG during NER in transiently transfected primary fibroblasts. The N-terminal PIP-UBM ubiquitin binding domain is more important for integrity of NER than the C-terminal PIP-box. I raise the model of an NER intermediate state that involves obligatory ubiquitination during NER and the blocking of error-prone translesion polymerases by XPG. This study excludes XPG as the responsible factor for PCNA recruitment and designates XPG as the factor as restrictive element for UV-damage dependent activation of translesion polymerases to S-phase. The results obtained with the endonuclease defective E791A XPG mutant confirm the actual “cut-patch-cat-patch” model of dual incision during NER. Moreover, this study clearly demonstrates the ability of endonuclease defective XPG to perform accurate NER in living cells. This accounts for the existence of a cellular backup mechanism for the XPG endonuclease function. The proposal for a nuclear backup mechanism is supported by the investigation of a physiologically relevant (evolutionary developed) XPG splicevariant with NER activity (IsoVI). The severely truncated XPG isoform is able to structurally complement a XPG defect. This complementation is dependent on the endonuclease function of Fen1. This suggests the existence of an evolutionary developed backup mechanism for XPG during NER

Anti-tumour compounds illudin S and Irofulven induce DNA lesions ignored by global repair and exclusively processed by transcription- and replication-coupled repair pathways.

Illudin S is a natural sesquiterpene drug with strong anti-tumour activity. Inside cells, unstable active metabolites of illudin cause the formation of as yet poorly characterised DNA lesions. In order to identify factors involved in their repair, we have performed a detailed genetic survey of repair-defective mutants for responses to the drug. We show that 90% of illudin's lethal effects in human fibroblasts can be prevented by an active nucleotide excision repair (NER) system. Core NER enzymes XPA, XPF, XPG, and TFIIH are essential for recovery. However, the presence of global NER initiators XPC, HR23A/HR23B and XPE is not required, whereas survival, repair and recovery from transcription inhibition critically depend on CSA, CSB and UVS, the factors specific for transcription-coupled NER. Base excision repair and non-homologous end-joining of DNA breaks do not play a major role in the processing of illudin lesions. However, active RAD18 is required for optimal cell survival, indicating that the lesions also block replication forks, eliciting post-replication-repair-like responses. However, the translesion-polymerase DNA pol eta is not involved. We conclude that illudin-induced lesions are exceptional in that they appear to be ignored by all of the known global repair systems, and can only be repaired when trapped in stalled replication or transcription complexes. We show that the semisynthetic illudin derivative hydroxymethylacylfulvene (HMAF, Irofulven), currently under clinical trial for anti-tumour therapy, acts via the same mechanism

DNA repair: Disorders

No description supplie

A New Disorder in UV-Induced Skin Cancer with Defective DNA Repair Distinct from Xeroderma Pigmentosum or Cockayne Syndrome

We report the characterization of a Japanese woman who exhibited many freckles and skin cancers in sun-exposed areas, but displayed no photosensitivity. Fibroblasts (KPSX7) derived from this patient showed similar UV sensitivity to that of normal human fibroblasts. The KPSX7 cells showed normal levels of unscheduled DNA synthesis, recovery of RNA synthesis, recovery of replicative DNA synthesis, protein-binding ability to UV-damaged DNA, and post-translational modification of xeroderma pigmentosum (XP) C. These results indicate that the patient had neither XP nor Cockayne syndrome. Although these results suggest that the KPSX7 cells were proficient in nucleotide excision repair activity, host-cell reactivation (HCR) activity of KPSX7 cells was reduced. Furthermore, introduction of UV damage endonuclease into the cells restored repair activity in the HCR assay to almost normal levels. These results indicate that KPSX7 cells are defective for some types of repair activity in UV-damaged DNA. In summary, the patient had a previously unknown disorder related to UV-induced carcinogenesis, with defective DNA repair

Unraveling DNA repair in human: molecular mechanisms and consequences of repair defect

Cellular genomes are vulnerable to an array of DNA-damaging agents, of both endogenous and environmental origin. Such damage occurs at a frequency too high to be compatible with life. As a result cell death and tissue degeneration, aging and cancer are caused. To avoid this and in order for the genome to be reproduced, these damages must be corrected efficiently by DNA repair mechanisms. Eukaryotic cells have multiple mechanisms for the repair of damaged DNA. These repair systems in humans protect the genome by repairing modified bases, DNA adducts, crosslinks and double-strand breaks. The lesions in DNA are eliminated by mechanisms such as direct reversal, base excision and nucleotide excision. The base excision repair eliminates single damaged-base residues by the action of specialized DNA glycosylases and AP endonucleases. Nucleotide excision repair excises damage within oligomers that are 25 to 32 nucleotides long. This repair utilizes many proteins to remove the major UV-induced photoproducts from DNA, as well as other types of modified nucleotides. Different DNA polymerases and ligases are utilized to complete the separate pathways. The double-strand breaks in DNA are repaired by mechanisms that involve DNA protein kinase and recombination proteins. The defect in one of the repair protein results in three rare recessive syndromes: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. This review describes the biochemistry of various repair processes and summarizes the clinical features and molecular mechanisms underlying these disorders

Xeroderma pigmentosum-variant patients from America, Europe, and Asia

Xeroderma pigmentosum-variant (XP-V) patients have sun sensitivity and increased skin cancer risk. Their cells have normal nucleotide excision repair, but have defects in the POLH gene encoding an error-prone polymerase, DNA polymerase eta (pol eta). To survey the molecular basis of XP-V worldwide, we measured pol eta protein in skin fibroblasts from putative XP-V patients (aged 8-66 years) from 10 families in North America, Turkey, Israel, Germany, and Korea. Pol eta was undetectable in cells from patients in eight families, whereas two showed faint bands. DNA sequencing identified 10 different POLH mutations. There were two splicing, one nonsense, five frameshift (3 deletion and 2 insertion), and two missense mutations. Nine of these mutations involved the catalytic domain. Although affected siblings had similar clinical features, the relation between the clinical features and the mutations was not clear. POLH mRNA levels were normal or reduced by 50% in three cell strains with undetectable levels of pol eta protein, indicating that nonsense-mediated message decay was limited. We found a wide spectrum of mutations in the POLH gene among XP-V patients in different countries, suggesting that many of these mutations arose independently.ope

Participation of mouse DNA polymerases iota, eta, and rev1 in translesion synthesis of carcinogen induced DNA adducts and carcinogenesis.

Recent advances in understanding the molecular mechanisms of mutagenesis indicate that most mutations are dependent on the activity of translesion synthesis DNA polymerases. The impact of reducing the level of these polymerases on mutagenesis and carcinogenesis in mouse models is poorly defined. Using knock out strategies we were able to remove polymerase eta (pol ç), and polymerase iota (pol é) and pol eta/pol iota from the mouse and lower REV1 in the mouse lung. This dissertation reports the changes in UV-induced carcinogenesis and mutagenesis that were observed. Mutagenesis data in pol iota deficient cells clearly indicate pol iota as a mutagenic TLS polymerase in UV lesion bypass. Pol iota removal effectively lowered the mutational frequency in both pol eta null and wild-type backgrounds. Unexpectedly, after Hprt mutant screening, pol eta and pol iota deficient cells were found to participate in UV lesion bypass in a strand-specific manner. This suggests that not only does bypass of photoproducts occur but occurs in an asymmetrical fashion, with preference of polymerases for leading or lagging strand. To examine the hypothesis that reducing the mutagenic load will reduce the incidence of cancer, we examined how pol iota status contributes to carcinogenesis. Despite the fact that pol iota was mutagenic in bypass of UV induced lesions, its removal accelerated cancer formation in the pol eta null background. The mechanisms behind this tumor suppressor function remain elusive, but indicate pol iota may have additional cellular roles in conjunction with its polymerase activity. Examining the hypothesis that reducing the mutagenic load will reduce the incidence of cancer, we developed strategies to reduce REV1 in mouse models of lung carcinogenesis. Endogenous REV1 mRNA in the lung was effectively lowered with the use of gene delivery of REV1 targeting ribozyme. This reduction was found to effectively decrease the multiplicity of B[a]P-induced lung tumors. This reduction did not affect the size or types of tumors induced suggesting inhibition of cancer formation occurred at the initiation step. Collectively, these data yield insight into the molecular mechanisms of mutagenesis that induce cancer formation