RNA polymerase II transcription suppresses nucleosomal modulation of UV-induced (6-4) photoproduct and cyclobutane pyrimidine dimer repair in yeast.

The nucleotide excision repair (NER) pathway is able to remove a wide variety of structurally unrelated lesions from DNA. NER operates throughout the genome, but the efficiencies of lesion removal are not the same for different genomic regions. Even within a single gene or DNA strand repair rates vary, and this intragenic heterogeneity is of considerable interest with respect to the mutagenic potential of carcinogens. In this study, we have analyzed the removal of the two major types of genotoxic DNA adducts induced by UV light, i.e., the pyrimidine (6-4)-pyrimidone photoproduct (6-4PP) and the cyclobutane pyrimidine dimer (CPD), from the Saccharomyces cerevisiae URA3 gene at nucleotide resolution. In contrast to the fast and uniform removal of CPDs from the transcribed strand, removal of lesions from the nontranscribed strand is generally less efficient and is modulated by the chromatin environment of the damage. Removal of 6-4PPs from nontranscribed sequences is also profoundly influenced by positioned nucleosomes, but this type of lesion is repaired at a much higher rate. Still, the transcribed strand is repaired preferentially, indicating that, as in the removal of CPDs, transcription-coupled repair predominates in the removal of 6-4PPs from transcribed DNA. The hypothesis that transcription machinery operates as the rate-determining damage recognition entity in transcription-coupled repair is supported by the observation that this pathway removes both types of UV photoproducts at equal rates without being profoundly influenced by the sequence or chromatin context.

De novo methylation as major event in the inactivation of transfected herpesvirus thymidine kinase genes in human cells.

Spontaneous inactivation of integrated thymidine kinase genes was studied in three human cell lines, one with multiple copies and two with a single copy of a transfected shuttle plasmid containing two selectable genes: the HSV tk gene and the Eco gpt gene. Selection for gpt expression prevented the isolation of TK- mutants which are the result of plasmid loss. Under these conditions TK- clones were isolated with a frequency of 5.10(-6) both with the cell line containing 5 or 6 copies of the tk gene and with one of the two cell lines containing one copy of this gene. This inactivity of the tk gene was associated with de novo methylation as the number of HAT-resistant (TK+) clones strongly increased after growth of the TK- derivatives in the presence of the demethylating agent, 5-azacytidine. Digestion with methylation-sensitive restriction enzymes revealed two different patterns of DNA methylation in the genomic DNA of TK- variants. In the TK- derivatives of the cell line containing multiple copies of the tk gene many HpaII restriction sites in the gene copies were insensitive to digestion. These HpaII sites were, however, not methylated in TK- variants of the cell line containing one copy of the plasmid, and methylated CpGs could be detected only with EcoRI which recognizes the cGAATTCg sequence in the tk promoter region. With the other of the two single-copy TK+ cell lines no TK- mutants were obtained, suggesting that the position of a gene in the genome is an important factor in determining the frequency and the extent of de novo methylation. Additionally, we observed that remethylation is an even more efficient process of gene inactivation as TK+ clones reactivated with 5-azacytidine can become TK- again at a 100-fold higher rate than the original TK+ cell line.

Defective Kin28, a subunit of yeast TFIIH, impairs transcription-coupled but not global genome nucleotide excision repair.

The essential Saccharomyces cerevisiae KIN28 gene encodes a subunit of general transcription factor TFIIH, a multiprotein complex required for RNA polymerase II transcription initiation and nucleotide excision repair (NER). Kin28 is implicated in the transition from transcription initiation to transcription elongation by phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of the RNA polymerase II complex. Here, we explore the possibility that Kin28 like the other subunits of TFIIH is involved in NER in vivo, using yeast cells carrying either a wildtype or a thermosensitive KIN28 allele. The removal of UV induced cyclobutane pyrimidine dimers (CPDs) was monitored at base resolution from both strands of the RNA polymerase II transcribed genes RPB2 and URA3. Cells carrying the thermosensitive KIN28 allele display a transcription-coupled repair (TCR) defect at the non-permissive temperature, which was most pronounced directly downstream of transcription initiation, probably as an indirect result of a general decrease in the level of RNA polymerase II transcription. The fact that CPD removal in non-transcribed DNA is completely unaffected in these cells indicates that Kin28 is not essential for general NER in vivo, providing the first example of a TFIIH subunit that is required for TCR but not for NER in general.

Studying DNA mutations in human cells with the use of an integrated HSV thymidine kinase target gene.

A shuttle vector carrying the origin of SV40 replication, the thymidine kinase (tk) gene of herpes simplex virus and the E. coli xanthine guanine phosphoribosyl transferase (gpt) gene has been introduced into human TK- cells. A transformed cell line containing only one stably integrated copy of the shuttle vector was used to study mutations in the introduced tk gene at the molecular level. Without selection for gpt expression, spontaneous TK- mutants arose at a frequency of approximately 10(-4)/generation, and were caused by deletion of plasmid sequences. However, when selection for expression of the gpt gene was applied, the background level of mutations at the tk gene was below 4.10(-6). From this cell line, TK- mutants were obtained after treatment with N-ethyl-N-nitrosourea (ENU). COS fusion appeared to be an efficient method for rescue and amplification of the integrated shuttle vector from the human chromosome. After further amplification and analysis in E. coli, rescued tk genes were easily identified and were shown to be physically unaltered by the rescue procedure. In contrast to rescued tk genes from TK+ cells, those obtained from the ENU-induced TK- mutants were unable to complement thymidine kinase-negative E. coli cells. Two such tk mutations were mapped in E. coli by marker rescue analysis. A GC----AT transition was the cause of both mutations. We show here that plasmid rescue by COS fusion is a reliable system for studying gene mutations in human cells, since no sequence changes occurred in rescued DNA except for the 2 ENU-induced sequence changes.

Lack of mammalian mutagenicity of the potent bacterial mutagen tris(2,3-dibromopropyl) phosphate and its metabolite 2-bromoacrolein.

The flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and its metabolite 2-bromoacrolein (2BA) are very potent bacterial mutagens in Salmonella typhimurium (S. typhimurium) TA 100. In this study, we showed that 2BA and Tris-BP are also mutagenic in S. typhimurium TA 104, which detects mutations at AT base pairs, while TA 100 detects mutations at CG basepairs. We also studied the mutagenicity of 2BA in mammalian cells in vitro and in the rat in vivo. Firstly, 2BA was tested in the human lymphoblastoid cell line TK6. The results showed that there was no increase in mutation frequency at the hprt locus, whereas there was a large decrease in cell survival. Secondly, a shuttle vector system was used to study the induction of mutations by 2BA:DNA adducts. The vector was modified by insertion of a single-stranded oligonucleotide containing on average one 2BA:DNA adduct. No increase in mutation frequency above background was detected after replication of this vector in SV40 transformed normal human fibroblasts. Because the liver is a major site for bioactivation of Tris-BP to 2BA in vivo, we tested the initiating capacity of Tris-BP in the rat liver in a modified Solt & Farber initiation and promotion system. Administration of Tris-BP resulted in a small increase in the number of preneoplastic gamma-glutamyl-transpeptidase positive (GGT+) foci in the liver compared to control animals (only significant in the lowest size class). Modification of the experimental protocol by performing partial hepatectomy 24 h after the administration of Tris-BP, did not increase the number of GGT+ or glutathione S-transferase-P (GST-P+) positive foci above the control level. Taken together, these results indicate that, in spite of a high mutagenicity in S. typhimurium, 2BA and Tris-BP have low or negligible mutagenic effects in mammalian systems. The lack of mutagenic activity may explain why Tris-BP is not a carcinogen in the rat liver.

Comparison of spontaneous hprt mutation spectra at the nucleotide sequence level in the endogenous hprt gene and five other genomic positions.

Mutation spectra at the nucleotide sequence level of five hprt cDNA genes integrated in different genomic positions of a HPRT(-) derivative of the human lymphoblastoid TK6 cell line were compared with each other and with the spectrum of mutations confined to the 657 bp coding region of the endogenous hprt gene in the parental TK6 cells. The mutation rates in these genomic positions vary significantly and also the mutation spectra are different. In each genomic position the majority of mutations are basepair substitutions and deletions. the ratios of which vary among the genomic positions. Although it is likely that the different rates of deletion are to a large extent the net result of different rates of misalignment and repair of these errors in the various genomic positions, for the basepair substitutions it is not possible to deduce which mechanisms have caused these mutations and what causes the differences among the genomic positions. Taken together, the differences in mutation rates and spectra cannot be explained by a single mutagenic process.

UV-induced mutagenesis in the endogenous hprt gene and in hprt cDNA genes integrated at different positions of the human genome.

The influence of the genomic position of a gene on UV-induced mutations was studied in the endogenous hprt gene in human lymphoblastoid TK6 cells and in cell lines derived from TK6 each containing a single copy of a hamster hprt cDNA gene integrated on a retroviral vector in different positions of the human genome. Previous studies showed that the genomic sequences surrounding the integration site influence spontaneous mutagenesis, resulting in a 10-fold difference in mutation rates among the hprt cDNA genes. Here we demonstrate that the genomic positions of three integrated hprt cDNA genes do not influence UV-induced mutagenesis. The mutability by UV irradiation in these cell lines is approximately the same (16.0 x 10(-6) per J/m2). The nature of the UV-induced mutations determined in two of the cell lines containing the integrated hprt cDNA gene (approximately 30 mutants each) was also found not to be different. The endogenous hprt gene in the parental TK6 cells exhibits a significantly lower mutability (2.1 x 10(-6) per J/m2) than the cDNA genes, but the spectrum is very similar. The spectrum in TK6 shows no influence of strand-specific repair and resembles most closely the spectrum obtained by McGregor et al. after irradiation of human cells synchronized in S-phase. This suggests that mutations arising in cells that are in S-phase at the time of irradiation constitute the majority of the mutants in an asynchronous TK6 cell population. We hypothesize that repair in the endogenous hprt gene in TK6 cells is very efficient, removing virtually all lesions before replication takes place except in cells that were in S-phase at the time of irradiation when there is not enough time for repair. Furthermore we suggest that the higher mutability of the integrated hprt cDNA genes compared with the endogenous gene is caused by a less efficient repair in the cDNA genes.

Gene amplification in a human osteosarcoma cell line results in the persistence of the original chromosome and the formation of translocation chromosomes.

Although gene amplification, a process that is markedly enhanced in tumor cells, has been studied in many different cell systems, there is still controversy about the mechanism(s) involved in this process. It is still unclear what happens to the DNA sequences that become amplified, whether they remain present at their original location (conservative gene amplification) or whether gene amplification necessarily results in a deletion at the original location (non-conservative gene amplification). We have studied gene amplification in a human osteosarcoma cell line, starting from a cell clone which contains only one copy of a plasmid integrate. Independent amplificants, originating from this clone and containing elevated plasmid copy numbers, were isolated and analyzed. Based on previous observations, encompassing the persistence of single-copy DNA sequences besides amplified DNA sequences clustered at a different location in the independent amplificants, we proposed an amplification pathway including a local duplication step and transposition of the duplicated DNA to other chromosomal positions. Now we have extended our study to more independent amplificants. We prove that the single-copy plasmid-containing chromosomes in the different amplificants and the single-copy plasmid-containing chromosome in the original parental cell clone are indeed identical, namely a translocation chromosome composed of at least three parts of which two originate from chromosomes 14 and 17. We show that the unit of amplification and the unit of the proposed transposition event are at least 1.5 Mb. We also demonstrate that the amplified DNA sequences, present at genomic locations other than the original single-copy DNA sequences, are preferentially associated with chromosome 16. We find that the amplified DNA sequences are often located at or near a site of chromosome translocation involving chromosome 16. In one cell clone we detect the amplified DNA sequences in most of the cells to be located within a complete chromosome 16 while in a minority of cells the amplified sequences are located at or near a breakpoint on a translocation chromosome 16. This indicates that this amplification region is highly unstable and frequently gives rise to translocation events.

Transcription-coupled and global genome repair in the Saccharomyces cerevisiae RPB2 gene at nucleotide resolution.

Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was examined at single nucleotide resolution in the yeast Saccharomyces cerevisiae, using an improved protocol for genomic end-labelling. To obtain the sensitivity required for adduct detection in yeast, an oligonucleotide-directed enrichment step was introduced into the current methodology developed for adduct detection in Escherichia coli. With this method, heterogeneous repair of CPDs within the RPB2 locus is observed. Individual CPDs positioned in the transcribed strand are removed very efficiently with identical kinetics. This fast repair starts within 23 bases downstream of the transcription initiation site. The non-transcribed strand of the active gene exhibits slow repair without detectable repair variations between individual lesions. In contrast, CPDs positioned in the promoter region show profound repair heterogeneity. Here, CPDs at specific sites are removed very quickly, with comparable rates to CPDs positioned in the transcribed strand, while at other positions lesions are not repaired at all during the period studied. Interestingly, the fast repair in the promoter region is dependent on the RAD7 and RAD16 genes, as are the slowly repaired CPDs in this region and in the non-transcribed strand. This indicates that the global genome repair pathway is not intrinsically slow and at specific positions can be as efficient as the transcription-coupled repair pathway.

Messenger ribonucleoprotein complexes in human KB cells infected with adenovirus type 5 contain tightly bound viral-coded '100K' proteins.

Late after infection of KB cells with adenovirus 5 an extra protein becomes associated with messenger ribonucleoprotein particles present in the polysomes. This protein has a molecular weight of 100000 and is identical to the virus coded '100K' protein found previously. The extra protein is firmly attached to the messenger ribonucleoprotein complexes. Its binding resists exposure to high salt concentrations as used in puromycin/high-salt dissociation and equilibrium centrifugation in Cs2SO4 gradients. In this respect it resembles the binding of two other proteins of Mr 74000 and 48000 which are commonly found in messenger ribonucleoprotein particles of various eukaryotic cells. The identity between the messenger ribonucleoprotein protein of Mr 100000 and the "100K' protein present in the soluble part of the cytoplasm was established by sodium dodecylsulphate/polyacrylamide gel electrophoresis, isoelectric focusing and peptide mapping after limited proteolysis with Staphylococcus aureus protease.

Transitions in the coupling of transcription and nucleotide excision repair within RNA polymerase II-transcribed genes of Saccharomyces cerevisiae.

The molecular mechanism of transcription-coupled nucleotide excision repair in eukaryotes is poorly understood. The identification of the dual role of basal transcription factor TFIIH in DNA repair and transcription provided a plausible link between both processes. However, TFIIH is not part of the elongating transcription complex, suggesting that additional components are required to recruit TFIIH when RNA polymerase II (RNAPII) stalls at the site of DNA damage. Previously, we have shown that the yeast Rad26 protein is involved in transcription-coupled DNA repair. This paper describes the differential contribution of the Rad26 protein to efficient removal of UV-induced cyclobutane pyrimidine dimers (CPDs) from transcribed DNA. Two distinct regions within the transcribed strand of RNAPII-transcribed genes are identified that differ in their requirement for the RAD26 gene product. Using high-resolution repair analysis, we determined the in vivo repair kinetics of cyclobutane pyrimidine dimers positioned around the transcription initiation site of RNAPII-transcribed genes RPB2 and URA3. Although transcription-coupled repair is severely reduced in rad26 mutants, lesions positioned in a small region immediately downstream of transcription initiation are efficiently removed in the absence of Rad26. The observed transition in repair characteristics is abrupt and in excellent agreement with the region where TFIIH dissociates from RNAPII in vitro, strongly suggesting an inverse correlation between TFIIH association and Rad26 requirement. These data suggest that a transcription repair coupling factor (Rad26/CSB) is required for efficient repair only during the elongating stages of RNAPII transcription.

Gene amplification in human cells may involve interchromosomal transposition and persistence of the original DNA region.

In tumor cells in vivo and in vitro the amplification of large DNA sequences is a spontaneous and frequently occurring genetic event. We have used human cells to study independent events leading to a low level of amplification of a single copy of an integrated plasmid. Fluorescence in situ hybridization, chromosome banding, and chromosome painting revealed that the new amplified DNA sequences can become located on chromosomes that are totally unrelated to the chromosome that harbors the original DNA sequences, indicating that the transposition of amplified DNA sequences is interchromosomal. In cells containing amplified DNA sequences the integrated single-copy plasmid remained at its original location. The unit of amplification contained a DNA fragment of at least a 800 kb and the same fragment was also present in the parental single-copy cell clone. The data suggest that a doubling of the DNA region at the original location precedes or is coupled to gene amplification.

Genome wide spontaneous mutation in human cells determined by the spectrum of mutations in hprt cDNA genes.

We have studied spontaneous mutagenesis in five hprt cDNA genes integrated at five different genomic positions in a human lymphoblastoid cell line (TK6). The spectra of 40 mutants from each position were combined to obtain a mutation spectrum of the overall genome. This collection of mutants was used to assess the contribution of several mutagenic processes to spontaneous mutagenesis. Deletions and single base pair changes account for the majority of the mutants and arise in approximately equal amounts (43 and 41%, respectively). The majority of the deletions and insertions are < 5 bp and are likely to be caused by template-directed misalignment (slippage) during replication. To account for frameshifts at non-iterated sites we propose a slightly different template-directed replication error model. A considerable amount of the observed base pair changes can also be explained by this last model, but several other processes leading to base pair changes such as depurination, deamination or spontaneously arising DNA damage are likely to contribute as well. We have compared this spectrum with mutation spectra in the endogenous hprt genes using published mutation data. It is shown that in the endogenous genes the contribution of base pair substitutions is much larger (71%) than in the hprt cDNA integrates and that deletions are less frequently observed (20%). The mutation rates of the integrated hprt cDNA genes show a mean increase of 30-fold as compared with the endogenous hprt gene. This results in a 60-fold increase of the absolute rate of deletion in the hprt cDNA genes and in a 15-fold increase of the base pair substitution rate. Replication errors such as slippage or the mechanism proposed in this study probably account to a large extent for this increase.

Spontaneous mutation spectrum in the hprt gene in human lymphoblastoid TK6 cells.

A spectrum of 100 mutations in the endogenous hprt gene of the human lymphoblastoid TK6 cell line is presented. The majority of the mutations originates in sequences outside the coding region of the gene. Large deletions are a major cause of inactivation of the hprt gene (57% of the mutants). Mutations in the splice sites that result in several forms of aberrantly spliced mRNA are relatively frequently recovered (16%) compared with mutants containing alterations in the coding region of the hprt gene (27%). The majority, but not all, of the splice mutants contain an alteration in the consensus sequences of the splice sites. A spectrum of mutations in the coding region of the hprt gene enlarged to a total of 42 mutants shows that basepair substitutions predominate (71%) and that small deletions and insertions are less frequently recovered. Basepair substitutions arise slightly more frequently at GC basepairs than at AT basepairs.

Genomic position influences spontaneous mutagenesis of an integrated retroviral vector containing the hprt cDNA as target for mutagenesis.

We have used five isogenic human lymphoblastoid cell lines each containing a retroviral vector at a different position in the genome to assess the influence of these positions on spontaneous mutagenesis. The vector contains the hamster hprt cDNA and the neo gene, both genes are transcribed from the retroviral LTR promoter. The rates of mutation leading to a HPRT- phenotype during growth in non-selective medium differed up to 60-fold in the five retroviral integrates, ranging from 5.9 x 10(-6) to 3.5 x 10(-4) mutations per cell generation. From each of the cell lines approximately 20 independent mutants were analyzed by Southern blot analysis. In two cell lines all mutations were caused by inactivation of the LTR promoter (presumably by DNA methylation), whereas in another cell line the estimated rate of this mutation is 1000-fold lower. Another important class of mutation is homologous recombination between the LTRs. This accounts for at least half of the mutants in the other three cell lines. Mutants carrying deletions or point mutations form a minor fraction of the mutant distribution. Mutations confined to the hprt cDNA sequences only were studied by selecting HPRT- mutants in the presence of G418. Even for this subset of mutations the rates can vary at least 10-fold between the different genomic positions, ranging from 4.2 x 10(-7) to 5.1 x 10(-6). We conclude therefore that mutations leading to a HPRT- phenotype are quantitatively as well as qualitatively different in the studied cell lines. This suggests that spontaneous mutagenesis in a gene is dependent on its position in the genome.

Mutation induction by UV light in retroviral hprt cDNA integrated at various chromosomal positions in repair-deficient hamster cells.

Mutation induction by UV irradiation was studied in a retroviral vector integrated in one copy per cell at various chromosomal positions. As a mutational target, hamster hprt cDNA was present on the retroviral vector. To minimize the influence of repair we used repair-deficient hamster cells, V-H1 and UV5, as a recipient for the vector. There is no major influence of chromosomal position on UV-induced mutation frequency and spectrum because no statistically significant difference between mutation induction in retroviral cDNA copies integrated at different chromosomal sites was observed. However, a major difference was found in mutation induction between the endogenous hamster hprt gene and the retroviral cDNA copies. Most noticeable was the absence in the cDNA of the strong strand bias for mutation induction, which was reported for the endogenous hprt gene. Our results with the hprt cDNA exclude as a general phenomenon a difference in mutation induction for leading and lagging strand DNA replication, which was proposed as an explanation for this strand bias in the endogenous gene. The similarity of mutation induction in the different retroviral cDNA copies, all directly surrounded by the same DNA sequence elements, together with the marked difference between the mutation induction in the endogenous gene and the cDNA copies may point to an important role of chromatin structure in mutation induction.