Understanding nucleotide excision repair and its roles in cancer and ageing.

Nucleotide excision repair (NER) consists of global genomic NER (GG-NER) and transcription coupled NER (TC-NER) subpathways. In eukaryotic cells, genomic DNA is wrapped around histone octamers (an H3–H4 tetramer and two H2A–H2B dimers) to form nucleosomes, which are well known to profoundly inhibit the access of NER proteins. Through unbiased screening of histone H4 residues in the nucleosomal LRS (loss of ribosomal DNA-silencing) domain, we identified 24 mutations that enhance or decrease UV sensitivity of Saccharomyces cerevisiae cells. The histone H4 H75E mutation, which is largely embedded in the nucleosome and interacts with histone H2B, significantly attenuates GG-NER and Rad26-independent TC-NER but does not affect TC-NER in the presence of Rad26. All the other histone H4 mutations, except for T73F and T73Y that mildly attenuate GG-NER, do not substantially affect GG-NER or TC-NER. The attenuation of GG-NER and Rad26-independent TC-NER by the H4H75E mutation is not due to decreased chromatin accessibility, impaired methylation of histone H3 K79 that is at the center of the LRS domain, or lowered expression of NER proteins. Instead, the attenuation is at least in part due to impaired recruitment of Rad4, the key lesion recognition and verification protein, to chromatin following induction of DNA lesions.

Platinum Resistance Related to a Functional NER Pathway

Nucleotide excision repair (NER) is a conserved DNA repair mechanism capable of removing a variety of helix-distorting lesions, such as UV-induced cyclobutane pyrimidine dimers (CPDs). NER can be grouped into two pathways: global genomic NER (GGR), which refers to repair throughout the genome, and transcription coupled NER (TCR), which refers to a repair mechanism that is dedicated to the transcribed strand (TS) of actively transcribed genes. In yeast S. cerevisiae, Rad7, Rad16, and Elc1 are specifically required for GGR. TCR is believed to be initiated by RNA polymerase II (Pol II) stalled at a lesion in the TS of a gene. Rad26, the yeast homolog of the human CSB protein, and RPB9, a nonessential subunit of Pol II, play important roles in TCR. However, the exact mechanisms of NER in eukaryotic cells are still elusive. By using yeast S. cerevisiae as a model organism, this dissertation focused on the functional mechanisms of transcription factor Tfb5, transcription elongation factors Spt4 and Spt5, and the putative yeast transcription repair coupling factor (TRCF) Rad26 in NER, especially in TCR pathway. Tfb5, the tenth subunit of the transcription/repair factor TFIIH, is implicated in one group of the human syndrome trichothiodystrophy (TTD). We found that Tfb5 plays different roles in different NER pathways in yeast. Tfb5 is essential for GGR and Rpb9 mediated TCR. However, Tfb5 is partially dispensable for Rad26 mediated TCR, especially in GGR deficient cells. Spt4 and its interacting partner Spt5 cooperatively suppress TCR only in the absence of Rad26, regardless of the presence of Rpb9. The phosphorylation of C-terminal repeat (CTR) domain of Spt5 by the Bur kinase plays an important role in the suppression. Immunoprecipitation results indicate that Rad26 dynamically associates with Pol II and restrains the binding of Spt4/Spt5 to Pol II. ATPase activity of Rad26 is required for facilitating TCR and for restraining the binding of Spt4/Spt5 to Pol II. Finally, we proposed that Rad26 enhances TCR by restraining the binding of suppressors Spt4/Spt5 to Pol II. These findings provide new insights into the functional mechanisms of Tfb5, Spt4/Spt5 and Rad26 in NER, especially in TCR.

Seventh Japanese-German workshop on molecular and cellular aspects of carcinogenesis

Mutations in cancer-associated genes result mainly from DNA-replication errors or endogenous and exogenous DNA lesions that escape the cellular DNA damage recognition and repair machinery. Dr. A. Yasui (Sendai, Japan) reported on the murine class I photolyases mCRY1 and mCRY2 and on the human DNA glycosylase hMYH (MutY homologue), which removes adenine from the post-replicative mismatch adenine:8-oxo-G. Recombinant mCRY1 and mCRY2 lack photolyase activity but may represent essential regulatory molecules determining circadian rhythms. Disruption of mCRY1 and mCRY2 in the germ line results in arrhythmia of the offspring's free running behavior in the dark. A novel cDNA, resulting from alternative splicing of hMYH, encodes a nuclear protein. cDNAs derived from either transcript suppress the Escherichia coli mutY mutator phenotype. Both hMYH proteins show glycosylase activity for adenine:8-oxo-G and binding to thymine or guanine paired with 8-oxo-G, without apparent catalysis. Further results suggest a role of hMYH in replication-coupled repair. As hMYH, human 8-oxo-G glycosylase hMMH (hOGG1) and thymine glycol glycosylase hNTH1 possess potential mitochondrial targeting sequences, stressing the significance of oxidative damage repair in both nuclear and mitochondrial DNA. Dr. S. Nishimura (Tsukuba, Japan), in collaboration with Dr. T. Noda (Tokyo, Japan), found that the type 1a isoform of endogenous hMMH exhibits 8-oxo-G glycosylase/AP lyase activity in human cells. hMMH type 1a is a major enzyme for repair of 8-oxo-G. Mmh–/– mice are physically normal; however, liver extracts lack nicking activity for 8-oxo-G containing substrate DNA, and the level of endogenous 8-oxo-G in liver DNA is 3-fold higher than in Mmh+/+ or Mmh+/– animals. In 14-week-old Mmh–/– mice, the level of 8-oxo-G was up to 7-fold higher, indicating accumulation with age. Ongoing studies must clarify whether or not Mmh–/– mice are more susceptible to mutation and cancer. Fjord region diol-epoxide metabolites of polycyclic aromatic hydrocarbons show higher tumorigenicity in rodents relative to the corresponding bay region diol-epoxides. Dr. H. Naegeli (Zurich, Switzerland) has tested whether this is due to differential repair of bulky DNA adducts formed by bay region benzo[a]pyrene (B[a]P) diol-epoxides vs. the structurally similar but more tumorigenic fjord region derivatives of benzo[c]phenanthrene (B[c]Ph). Stereospecific B[a]P and B[c]Ph adducts were synthesized on adenine-N6 at the second position of codon 61 of human ras, a known hot-spot for carcinogen-induced ras activation. When incubated with human cell extracts, all tested bay region B[a]P-N6-dA adducts were removed by nucleotide excision repair (NER) with varying efficiency, whereas all fjord region B[c]Ph-N6-dA adducts were refractory to NER. The pronounced tumorigenicity of B[c]Ph-derived and -related fjord region diol-epoxides may thus be due, at least in part, to inefficient repair of their stable base adducts formed in DNA. Dr. G.T.J. van der Horst (Rotterdam, The Netherlands) discussed the consequences of defective global genome NER (GG-NER) and transcription-coupled NER (TC-NER) in terms of human predisposition to cancer, neurological diseases, and other abnormalities. Most Xeroderma pigmentosum (XP) patients exhibit defective GG-NER and TC-NER. However, only GG-NER is affected in XP complementation group C (XPC) and only TC-NER is impaired in Cockayne syndrome (CS). Contrary to XP, CS and Trichothiodystrophy (TTD) are not associated with skin cancer predisposition. Using transgene and gene knockout methodology, NER disorders can be mimicked in mice. The synergy of genes can be studied in double mutants, and marker genes can be added to the genome. XPA–/– mice are entirely NER-negative and rapidly develop skin tumors upon exposure to UV or dimethylbenz[a]anthracene. In aging XPA animals, internal cancers occur spontaneously or are readily inducible by B[a]P. GG-NER-defective XPC mice also exhibit increased susceptibility to UV-induced skin cancer, as do CSB (TC-NER-deficient) and XPDTTD mice (25% residual NER activity). In rodents, therefore, any interference with the NER machinery results in increased cancer susceptibility. Unlike XPA mice, animals deficient in the ERCC1 protein (normally complexed with XPF and responsible for the 5′ incision in NER) exhibit embryonal or early post-natal lethality due to severe liver and kidney dysfunction. ERCC1 must, therefore, have an additional function connected with NER. Moreover, ERCC1 may be involved in the repair of DNA interstrand cross-links by mitotic recombination. Mouse models have thus become valuable tools for analysis of the contributions of GG-NER and TC-NER to the prevention of cytotoxic, mutagenic, and oncogenic effects of DNA-reactive carcinogens. They may also help us to understand some of the clinical symptoms in human disorders, including aging. A thus far unknown mode of DNA repair observed in N-alkyl-N-nitrosourea–induced rat mammary carcinogenesis was described by Dr. J. Thomale (Essen, Germany). This repair mechanism strongly affects gene-specific mutation frequencies by selectively and efficiently eliminating miscoding O6-ethyl-, but not O6-methylguanines from both DNA strands of transcriptionally active genes, independent of the repair of these lesions by O6-methylguanine-DNA methyltransferase. In a large-scale program performed at the GSF National Research Center for Environment and Health near Munich, mouse mutants were induced by N-ethyl-N-nitrosourea (EtNU). This “phenotype-driven” approach to the identification of mutants relevant to the pathogenesis of human diseases is modeled after the genetic dissection of body pattern formation in Drosophila. As described by Dr. R. Balling (Neuherberg, Germany), the intention is to identify the responsible genes or defects through positional cloning and other strategies. Functional analyses will require multiple alleles of the same gene or different genes involved in the pathogenesis of the same disease, including hypomorphs, alleles of different strengths, and gain-of-function alleles. Offspring of the F1 and F3 generations of EtNU-treated mice are analyzed for dominant and recessive phenotypic abnormalities. About 18,000 mice have been screened within the last 2 years, and 170 new mouse mutants have already been isolated. Dr. H.H. Grunicke (Innsbruck, Austria) has studied the involvement of protein kinase C (PKC) isoforms λ and ζ in the Ras-mediated reorganization of the actin cytoskeleton. Transforming Ha-Ras L61 causes profound alterations in NIH 3T3 cells, including the disassembly of actin stress fibers. This effect is abrogated by co-expression of kinase-defective, dominant-negative mutants of aPKC-λ and aPKC-ζ as well as by anti-sense constructs directed against isotype-specific 5′ sequences of the corresponding mRNA but not by co-expression of a kinase-defective dominant-negative mutant of cPKC-α. Constitutively active mutants of PKC λ and ζ mimic the cytoskeletal effect of Ras. PKC-λ may, therefore, act upstream whereas PKC-ζ may function downstream of Rac-1 in the signaling from Ha-Ras to the cytoskeleton. Thus, co-transfection of plasmids encoding Ha-Ras L61 and either PKC-λ or PKC-ζ enhances the kinase activity of both enzymes, and Ras-mediated activation of PKC-ζ is abrogated by co-expression of dominant-negative Rac-1 N17. The Ras-Rac-Rho pathway apparently co-operates with the Ras-Raf-ERK cascade in transcriptional activation of the cyclin D1 promoter. Dominant-negative PKC-ζ inhibits activation of a cyclin D1-luciferase reporter by constitutively active Raf, implicating the participation of PKC-ζ in the Ras-Raf-ERK pathway. PKC-λ regulates Ras-mediated activation of cyclin D1 by a Raf-ERK–independent mechanism. Activation of PKC-λ by Ras appears to activate an essential survival pathway involving Akt and counteracting Ras-induced apoptosis. Using human epidermal keratinocytes, Dr. T. Kuroki (Tokyo, Japan) has studied PKC as a mediator of signals for squamous-cell differentiation. Among 10 PKC isoforms, PKC-η plays a crucial role. It is activated by cholesterol sulfate, which up-regulates the synthesis of membrane-bound transglutaminase 1, a key enzyme in squamous-cell differentiation. Terminal differentiation is accompanied by G1 arrest of cells and apparently involves a direct association of PKC-η molecules with the cell-cycle regulatory machinery. In organisms ranging from nematodes to mammals, the Wnt/Wingless signaling pathway [which includes Frizzled (Wnt receptor), Dvl, GSK-3β (protein kinase), β-catenin, and Tcf/Lef (transcription factor)] is of key importance in development; in the control of cell proliferation, motility, and fate; and in the subversion of these processes in carcinogenesis. As described by Drs. A. Kikuchi (Hiroshima, Japan) and T. Akiyama (Tokyo, Japan), Wnt signaling involves the accumulation of β-catenin, a positive regulator that associates with Tcf/Lef, resulting in expression of target genes (e.g., c-myc, c-jun, fra, cyclin D1). The tumor-suppressor APC induces degradation of β-catenin. Axin, a negative regulator, binds directly to glycogen synthase kinase-3β (GSK-3β), β-catenin, and APC and enhances GSK-3β-dependent phosphorylation of β-catenin, the β-catenin homologue plakoglobin, APC, and Axin itself. Dvl and protein phosphatase 2A bind to Axin, inhibiting its phosphorylating ability. Over-expression of Axin suppresses cell proliferation, Wnt-3a-mediated β-catenin accumulation, and Tcf activation. An APC fragment inducing β-catenin degradation was rendered inactive by disruption of its Axin-binding sites. Moreover, a fragment spanning the regulation of G-protein signaling (RGS) domain of Axin inhibited APC-mediated β-catenin degradation, as did an APC fragment with mutated β-catenin but intact Axin-binding sites. Axin may thus be a scaffold protein selectively channeling the signal from Wnt to β-catenin and plakoglobin. Drs. J. Schnekenburger and M.M. Lerch (Münster, Germany) showed that the integrity of adherens junctions between pancreatic acinar cells is maintained by proteins of the E-cadherin–β-catenin complex. During the formation, maintenance, or restitution of intercellular contacts, the phosphotyrosine phosphatases (PTPs) SHP-1 and κ are endogenously expressed. Their disruption is paralleled by tyrosine phosphorylation of adhesion proteins and by the dissociation of PTP κ from the cadherin–catenin complex. In contrast, the cytoplasmic PTP SHP-1 associates with tyrosine-phosphorylated proteins of the dissociated, inactive complex, suggesting involvement in its dephosphorylation and reconstitution. Maintenance of the cadherin–catenin complex and regulation of intercellular contacts in the pancreas thus depend on the activity of PTPs κ and SHP-1, and PTPs may represent a novel class of tumor suppressors. Observations on phosphotyrosine-mediated signaling shed new light on the process of receptor tyrosine kinase (RTK) activation and signal definition. Extending established mechanisms of ligand-induced homodimeric receptor complex formation, these findings highlight heterodimeric receptor aggregation as a powerful means of signal diversification and expansion. Dr. A. Ullrich (Martinsried, Germany) reported that promiscuous receptor interactions involve different ligand-binding kinetics and generate divergent receptor phosphorylation sites, which could allow enhanced or modified signaling. A newly defined RTK function involves signal integration from a variety of stimuli, including calcium-dependent responses in neuronal cells, activation of G protein–coupled receptors, or cellular stress. RTKs must be considered critical foci and switch points for multiple environmental and internal stimuli. A novel mechanism of phosphotyrosine signal generation involves constitutively active rather than inducible tyrosine kinases. These are in a dynamic equilibrium with constitutively active PTPs. RTK activation then occurs by inactivation of PTPs through oxidation or physical disruption of RTK–PTP interactions. Ceramide produced by sphingomyelinases (SMases) has been recognized as a second-messenger in membrane receptor and possibly apoptotic signaling. Dr. M. Krönke (Cologne, Germany) described 2 distinct types of SMase activated by TNF via binding to distinct cytoplasmic domains of the p55 TNF receptor (TNF-R55): a membrane-associated neutral (N-)SMase and an endosomal acid (A-)SMase. N-SMase activation is mediated by the WD-repeat protein FAN, which binds to a motif N-terminally adjacent to the death domain of TNF-R55. A-SMase activation is signaled by the TNF-R55 death domain through the adaptor proteins TRADD and FADD. Each of these SMases couples to a select, non-overlapping signaling cascade. Downstream targets of ceramide produced by either SMase have not been defined unequivocally. However, N-SMase-derived ceramide may couple to a ceramide-activated membrane protein kinase (CAPK), which in turn phosphorylates and activates Raf-1 kinase. A-SMase–derived ceramide specifically binds to and activates cathepsin D. Ceramide does not physically interact with apoptotic caspases, and A-SMase has not been identified as a target for caspase cleavage and/or activation. The postulated connection between A-SMase, ceramide, and apoptosis may thus reside in cathepsin D. Comprising IL-6, IL-11, leukemia inhibitory factor, oncostatin M (OSM), ciliary neurotrophic factor, and cardiotrophin-1 and signaling through their common receptor subunit gp130, “IL-6-type cytokines” play an important role in the regulation of gene activation, cell proliferation, and differentiation. As reported by Dr. P.C. Heinrich (Aachen, Germany), IL-6 cytokines cause proliferation arrest in human A375 melanoma cells. This effect depends on the activation of STAT transcription factors: (i) the intensity of STAT3 and STAT1 signals is correlated with the extent of inhibition exerted by IL-6, IL-6 plus soluble IL-6 receptor, or OSM; (ii) truncated chimeric receptor retaining only the membrane-proximal region of gp130 neither activates STATs nor mediates proliferation arrest in stable transfectants, functions that are restored by addition of short STAT recruitment modules comprising critical tyrosine residues of gp130 (Y767, Y814), and a receptor carrying tyrosine module Y759 of gp130 effectively mediates activation of phosphatase SHP-2 but does not affect cell proliferation; (iii) over-expression of dominant-negative forms of STAT3, but not STAT1, abrogates the inhibitory effect of OSM and IL-6. The cyclin-dependent kinase inhibitor p27KIP1 was identified as a novel target regulated by IL-6 cytokines. Up-regulation of p27KIP1 mRNA was STAT3-dependent, and p27KIP1 accumulated in 3 human melanoma cell lines sensitive to IL-6 cytokines, coinciding with the disappearance of hyperphosphorylated retinoblastoma protein. Dr. T. Möröy (Essen, Germany) has tested cyclin E for its oncogenic activity in T-cell lymphomagenesis using transgenic mice expressing high levels of cyclin E in their T lymphocytes. These animals were neither predisposed to spontaneous tumorigenesis nor did they exhibit increased tumor incidence when cross-bred with Eμ L-myc transgenic mice; however, they developed hyperplasia of peripheral lymphoid organs at a later age (incidence approx. 27%). When exposed to N-methyl-N-nitrosourea (MeNU), transgenic mice developed T-cell tumors at higher incidence (approx. 54%) than their non-transgenic counterparts (31%). In the latter, 1/8 lymphomas carried an activating Ki-ras mutation, as opposed to 5/16 lymphomas of the transgenics. Cyclin E over-expression per se did not result in higher CDK2 activity; however, all tumors of MeNU-treated normal and transgenic mice exhibited down-regulation of p27KIP1 and increased histone H1 kinase activity. High-level expression of cyclin E can, therefore, predispose murine T cells to hyperplasia and malignant conversion, but this requires co-operation with other activated oncogenes. Upon DNA damage, TP53 accumulates rapidly in the target cells and is activated as a transcription factor, resulting in proliferation arrest or apoptosis. The relationship between phosphorylation of human TP53 and the regulation of its activity was investigated by Dr. Y. Taya (Tokyo, Japan) with the use of antibodies recognizing most of the potential TP53 phosphorylation and acetylation sites. Among these are novel phosphorylation sites at the transcriptional activation domain 43–63 and the proline-rich domain 64–92, deletion of either of which abolishes the induction of apoptosis. Contrasting with Ser15 and Ser20 of the MDM2 binding domain, phosphorylation of Ser46 and Ser90 is induced only at a late stage, after exposure to DNA-reactive agents. The kinetics of phosphorylation at these sites and of the induction of apoptosis are similar. Phosphorylation at Ser46 and Ser90 may thus control the transcriptional activation of apoptosis-inducing genes by TP53. Most, if not all, prokaryotes maintain their genomes in the form of circular DNAs; however, the genomic DNA of all eukaryotes is linear. As pointed out by Dr. F. Ishikawa (Yokohama, Japan), a linear genome implies the presence of DNA termini (telomeres). DNA can easily undergo end-to-end fusion. However, the linear DNA molecules present as chromosomes in eukaryotes never react in this way because telomeres protect DNA termini from fusion. Conventional DNA replication does not occur at the very ends of telomeres, and as cells proliferate, this “end replication deficiency” results in the gradual shortening of telomeres. A specialized reverse transcriptase, telomerase, can synthesize telomeric DNA and thereby counteract telomeric erosion. Contrary to many of their malignant counterparts, most normal cells do not express telomerase. Telomeres thus shorten with age. What benefit could be obtained from the extra cost required to maintain the stability of a linear genome? From fission yeast mutants defective in 2 ATM which due to of telomeric DNA, cells that maintain their genomes in the form of circular chromosomes were for the These cells were to undergo suggesting a between and Dr. Germany) described early lesions induced in rodents and for similar alterations in the of human the of 3 cell can be (i) the (ii) the and (iii) the distinct are at the (i) with of glycogen accompanied by of and signaling indicating effects of and (ii) in glycogen but in in with an enzyme pattern of a The is by an activated tyrosine with over-expression of the receptor, receptor, and produced in by of pancreatic cells, results in which to later A from to occurs at more of The exhibits increased of and mitochondrial and This effect is also observed in of that the and cell may The rat of predisposition to cell was discussed by Dr. (Tokyo, A mutation in the rat homologue of the human gene the exposed to develop not However, which are for the mutation to with high develop but not tumors from as early as The mutation thus the of required and the tumor indicating that can to both tumors and of of the was in the lesions and appears to be the indicating a tumor-suppressor function of proliferation of lesions that critical including required for the to and on a carcinogenesis does not in a the rat an for the analysis of in the genes result in between and within the same The identification of genes in other models may help us to understand the phenotypic in cancer is a major cause of death in A mutation of the gene is responsible for cancers of with very high However, genes associated with cancers of the type are Using the rat of carcinogenesis induced by Dr. T. (Tokyo, Japan) found that genes are involved in the differential effect in vs. A gene on cancer susceptibility gene cancer incidence by the genes, cancer gene on and on suppress cancer on the and of tumor The rat of and to human chromosomes and A mutation at nucleotide in the region of the gene on rat is for induced by exposure to on post-natal develop of the with an incidence of the are entirely Dr. A. (Essen, Germany) reported that was strongly in EtNU-treated F1 animals incidence approx. analyses and deletion in F1 showed that the is the of with of 10 of The region of this is on the telomeric of analysis of and animals, was to a region from of F1 tumors exhibited additional in this mutant cells are present at in the of both and animals. However, their in and these cells are by the exhibit of and malignant The of thus appears to involve mechanisms the recognition and of cells. are the most common malignant disorders in by Dr. Germany) suggest the involvement of control genes in and to the of 2 including were observed on chromosomes (approx. of and of and of genetic on was observed in of with a common region of at The tumor-suppressor gene on is and/or in a high of of the gene at occur in mainly of the is the human homologue of the which encodes a receptor regulated by of the This pathway plays an important role in analyses of and defined cell have identified 2 distinct the clinical of which are is by an has a and from cells in the cell The second involves alterations of and apparently from cells of the has activity. Dr. H. (Tokyo, Japan) has investigated its effect in the rat of carcinogenesis induced by by a or for in the incidence of were observed and of In mice, for the of at the of In a rat of carcinogen-induced of for in a of and a of In mice carrying of cancer cells, the of foci in the of and higher of of and cells of were observed in and of the of Ras may have for the of The key of Ras as a signal in its to switch between the active and inactive of this by of ras results in tumorigenesis in As described by Dr. A. Germany), of Ras is very but is by the activating protein which an the active The positive of the contacts the and the The activity of oncogenic ras mutants is not enhanced by but can, in be thus a valuable in the of to the of cancer cells with the use of proteins were discussed by Dr. Germany). and/or over-expression of the gene is observed in types of human including mammary and and with an of the function of directly affects tumor cell The of cells expressing is by dominant-negative receptor of by tumor cells via receptor or ligand-induced can be through molecules binding selectively to the domain of derivatives were to as well as an protein T cells to tumor cells and to via have new mechanisms of that may to the process of carcinogenesis. As pointed out by Dr. M. the global of is the of which depends on genetic and the of at of the or and tumor thus and is for the of multiple as required for the expression of malignant via genetic accompanied by the of due to the accumulation of and may become and later This can be that many that been A for cancer could thus be on the of malignant cells. These cells have to multiple and are to to new (e.g., However, they may have many of the functions they new are to the both and to to a on the of apoptosis Y. Japan) and on mouse mutants as novel models for human diseases as well as identification of the cells of of on genetic differentiation of their Germany). The of the on and of have been by the for of the for the for of the of Health and and the Research of the of Research and on the by the of Research and and the and by other from both

The role of Cockayne syndrome group A (CSA) protein in transcription-coupled nucleotide excision repair

Tfb5 is partially dispensable for Rad26 mediated transcription coupled nucleotide excision repair in yeast

UV light causes the formation of pyrimidine dimers (PDs). Transcription-coupled (TC) nucleotide excision repair (NER) and global genome (GG) NER remove PDs from the transcribed strand (TS) of active genes and the inactive genome, respectively. TC-NER is triggered by elongating RNA polymerases that are blocked at PDs. The yeast rRNA genes are densely loaded with RNA polymerase-I. After UV irradiation, their density increases at the 5'-end of the gene, which results from continuous transcription initiation, followed by elongation and pausing/release at the first encountered PD, from the transcription start site. RNA polymerase-I posed at downstream PDs are released from the TS and are replaced by nucleosomes. Consequently, discrete chromatin structures are formed in the damaged transcribed rRNA genes. Singular assignation of the two NER sub-pathways could therefore be required to eliminate PDs from the TS. To advance our understanding of NER in the dynamic structure of transcribed chromatin, we investigated the repair of PDs at nucleotide resolution in separate rRNA gene coding regions. In the TS, the TC-NER efficiency reflected the density of RNA polymerase-I, and PDs were removed faster in the 5'-end than in the 3'-end of the gene. GG-NER removed PDs from the TS where RNA polymerase-I was transiently replaced by a nucleosome. The two NER sub-pathways inversely participated to remove PDs from the TS. In the non-TS of both nucleosome and non-nucleosome rRNA gene coding regions, GG-NER was solely responsible to remove UV-induced DNA lesions.

Nucleotide excision repair (NER) is carried out by xeroderma pigmentosum (XP) factors. Before the excision reaction, DNA damage is recognized by a complex originally thought to contain the XP group C responsible gene product (XPC) and the human homologue of Rad23 B (HR23B). Here, we show that centrin 2/caltractin 1 (CEN2) is also a component of the XPC repair complex. We demonstrate that nearly all XPC complexes contain CEN2, that CEN2 interacts directly with XPC, and that CEN2, in cooperation with HR23B, stabilizes XPC, which stimulates XPC NER activity in vitro. CEN2 has been shown to play an important role in centrosome duplication. Thus, those findings suggest that the XPC-CEN2 interaction may reflect coupling of cell division and NER.

Cyclobutane pyrimidine dimers (CPDs) are predominant ultraviolet (UV) light-induced DNA lesions that can result in mutations and lead to skin cancers (1). CPD lesions are primarily repaired by nucleotide excision repair (NER), a highly conserved repair pathway (2). NER consists of two subpathways: transcription-coupled NER (TC-NER) and global genome NER (GG-NER) (2). GG-NER can occur anywhere in the genome, whereas TC-NER is dedicated for repair of bulky lesions at transcribed strands of active genes (2). Eukaryotic TC-NER is initiated by stalled RNA polymerase II (Pol II) at DNA damage sites, whereas the damage recognition in GG-NER DNA is mainly achieved by repair factor XPC (2). Many factors can modulate the repair efficiency and outcome in vivo, including the chromatin organization and genomic location of lesions, accessibility of repair factors, and occupancy of repair facilitators and suppressors. In PNAS, Duan et al. (3) take a genomic approach to investigate how the genome-wide contribution of several key protein factors would impact the TC-NER process of CPDs in yeast, providing important insights into how TC-NER is modulated in yeast chromatin. In particular, Duan et al. (3) focus on three factors, Rad26, transcription factor IIH (TFIIH), and elongation factor Spt4/Spt5, that have dual roles in both TC-NER and transcription processes. During TC-NER, human Cockayne syndrome group B (CSB) protein and its yeast ortholog Rad26 play important roles in TC-NER. CSB/Rad26 is the first protein to be recruited to DNA damage-stalled Pol II (2). Human CSB, CSA, and UVSSA act cooperatively to recruit TFIIH to damage-stalled Pol II (4). TFIIH is involved in the lesion verification step as well as recruitment of repair factors for dual incision (2, 5). In contrast, yeast Spt4/Spt5 functions as a TC-NER suppressor and Rad26 is required to antagonize Spt4/Spt5’s suppression (6). Recent structure of the Pol II–Rad26 complex … [↵][1]1Email: dongwang{at}ucsd.edu. [1]: #xref-corresp-1-1

The effects of UV irradiation on herpes simplex virus type 1 (HSV-1) gene expression and DNA replication were examined in cell lines containing mutations inactivating the XPA gene product required for nucleotide-excision repair, the DNA polymerase eta responsible for translesion synthesis, or the Cockayne syndrome A and B (CSA and CSB) gene products required for transcription-coupled nucleotide excision repair. In the absence of XPA and CSA and CSB gene products, virus replication was reduced 10(6)-, 400-, and 100-fold, respectively. In DNA polymerase eta mutant cells HSV-1 plaque efficiency was reduced 10(4)-fold. Furthermore, DNA polymerase eta was strictly required for virus replication at low multiplicities of infection but dispensable at high multiplicities of infection. Knock down of Rad 51, Rad 52, and Rad 54 levels by RNA interference reduced replication of UV-irradiated HSV-1 150-, 100-, and 50-fold, respectively. We find that transcription-coupled repair efficiently supports expression of immediate early and early genes from UV-irradiated HSV-1 DNA. In contrast, the progression of the replication fork appears to be impaired, causing a severe reduction of late gene expression. Since the HSV-1 replisome does not make use of proliferating cell nuclear antigen, we attribute the replication defect to an inability to perform proliferating cell nuclear antigen-dependent translesion synthesis by polymerase switching at the fork. Instead, DNA polymerase eta may act during postreplication gap filling. Homologous recombination, finally, might restore the physical and genetic integrity of the virus chromosome.

From its very beginning, life has faced the fundamental problem that the form in which genetic information is stored is not chemically inert. DNA integrity is challenged by the damaging effect of numerous chemical and physical agents, compromizing its function. To protect this Achilles heel, an intricate network of DNA repair systems has evolved early in evolution. One of these is nucleotide excision repair (NER), a highly versatile and sophisticated DNA damage removal pathway that counteracts the deleterious effects of a multitude of DNA lesions, including major types of damage induced by environmental sources. The most relevant lesions subject to NER are cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs), two major kinds of injury produced by the shortwave UV component of sunlight. In addition, numerous bulky chemical adducts are eliminated by this process. Within the divergent spectrum of NER lesions, significant distortion of the DNA helix appears to be a common denominator. Defects in NER underlie the extreme photosensitivity and predisposition to skin cancer observed with the prototype repair syndrome xeroderma pigmentosum (XP). Seven XP complementation groups have been identified, representing distinct repair genes XPA–G (discussed in detail below). In the last decade, all key NER factors have been cloned and the core of the ‘cut-and-paste’ reaction has been reconstituted in vitro from purified components. Recently, XPC (complexed to hHR23B) has been identified as a DNA-damage sensor and repair-recruitment factor. The general transcription factor complex TFIIH, containing the XPB and XPD helicases, mediates strand separation at the site of the lesion. XPA verifies the damage in an open DNA conformation and is crucial in the assembly of the remainder of the repair machinery. Replication protein A (RPA) stabilizes the opened DNA complex and is involved in positioning the XPG and ERCC1–XPF endonucleases responsible for the DNA incisions around the lesion. After removal of the damagecontaining oligonucleotide, typically 24–32 nucleotides in length, general replication factors fill in the remaining gap and close it. Two modes of NER can be distinguished: repair of lesions over the entire genome, referred to as global genome NER (GG–NER), and repair of transcription-blocking lesions present in transcribed DNA strands, hence called transcription-coupled NER (TC–NER). Most XP groups harbor defects in a common component of both NER subpathways. GG–NER is dependent on the activity of all factors mentioned above, including the GG– NER-specific complex XPC–hHR23B. The rate of repair for GG–NER strongly depends on the type of lesion. For instance, 6-4PPs are removed much faster from the genome than CPDs, probably because of differences in affinity of the damage sensor XPC–hHR23B. In addition, the location (accessibility) of a lesion influences the removal rate in vivo. In TC–NER, damage is detected by the elongating RNA polymerase II complex when it encounters a lesion. Interestingly, a distinct disorder, Cockayne syndrome (CS), is associated with a specific defect in transcription-coupled repair. The identification of two complementation groups (CS-A and CS-B) shows that at least two gene products are specifically needed for fast and efficient repair of transcribed strands. Phenotypically, CS is a very pleiotropic condition characterized by photosensitivity as well as severe neurological, developmental, and premature aging features. Most of these symptoms are not seen even with totally NERdeficient XP patients. The additional symptoms of CS suggest that transcription-coupled repair and/or the CS proteins have functions beyond NER. Also, non-NERspecific lesions (such as oxidative damage) that stall transcription elongation appear to be removed in a transcription-coupled fashion, linking a blocked polymerase to multiple repair pathways. Intriguingly, some XP-B, XP-D, and XP-G patients display CS features combined with XP manifestations. Yet other XP-B and XP-D individuals suffer from the CS-like brittle-hair syndrome trichothiodystrophy (TTD). This clinical conundrum points to additional roles of these NER factors as well. A recent mouse model for TTD has linked mutations in the XPD subunit of the dual functional TFIIH complex with deficiencies in basal transcription underlying at least some of the TTD manifestations. Thus, NER defects are associated with a surprisingly wide clinical heterogeneity due to additional functions of the NER factors involved. 1Corresponding author. E-MAIL Hoeijmakers@gen.fgg.eur.nl; FAX 31 10 408 9468.

Nucleotide excision repair (NER) is one of the most important DNA repair systems involved in removing a wide range of DNA damage from the genome. NER consists of two sub-pathways: the global genome nucleotide excision repair (GG-NER) pathway, which removes DNA lesions generated in the whole genome (as described in Chap. 1 of this book), and the transcription-coupled nucleotide excision repair (TC-NER) pathway, which removes lesions specifically from the transcribed strands of actively transcribed genes. At least 20 factors are involved in the TC-NER process, and mutations in the genes responsible for coding these factors may mainly result in two human genetic disorders: Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS). Despite similar molecular defects in TC-NER, CS and UVSS show distinct clinical phenotypes. CS patients display severe developmental and neurological abnormalities as well as premature ageing, whereas UVSS individuals only show milder cutaneous abnormalities, such as hypersensitivity to UV light. The molecular basis for the difference in the clinical features remains unclear. In this chapter, we will specifically describe the historical progress and recent findings of TC-NER and summarize the current understanding of the molecular pathogenesis of CS and UVSS.

The NER proteins XPC and CSB, but not ERCC1, regulate the sensitivity to the novel DNA binder S23906: Implications for recognition and repair of antitumor alkylators

UV irradiation is one of the major external insults to cells and can cause skin aging and cancer. In response to UV light-induced DNA damage, the nucleotide excision repair (NER) pathways are activated to remove DNA lesions. We report here that testicular nuclear receptor 4 (TR4), a member of the nuclear receptor family, modulates DNA repair specifically through the transcription-coupled (TC) NER pathway but not the global genomic NER pathway. The level of Cockayne syndrome B protein (CSB), a member of the TC-NER pathway, is 10-fold reduced in TR4-deficient mouse tissues, and TR4 directly regulates CSB at the transcriptional level. Moreover, restored CSB expression rescues UV hypersensitivity of TR4-deficient cells. Together, these results indicate that TR4 modulates UV sensitivity by promoting the TC-NER DNA repair pathway through transcriptional regulation of CSB. These results may lead to the development of new treatments for UV light-sensitive syndromes, skin cancer, and aging.