Judith G. Tasseron-de Jong

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

ABSTRACT 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.

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.

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.

Cytosine methylation in the EcoRI site of active and inactive herpesvirus thymidine kinase promoters

The herpesvirus thymidine kinase (tk) gene integrated in the human cell line, 2.1-a, can be inactivated by limited de novo methylation. All these TK- clones show partial EcoRI digestion of the recognition site (cGAATTCg) in the tk promoter in contrast to complete digestion of this site in the original cell line. Studies on well-defined substrates prepared in vitro showed that methylation of one cytosine in the EcoRI recognition sequence resulted in partial and methylation of both cytosines in severe inhibition of digestion by EcoRI. This characteristic was used to determine whether no, one or both cytosines in the EcoRI site of the tk promoter were methylated in various TK- clones derived from 2.1-a and in TK+ clones re-expressing the gene after 5-azacytidine treatment. A high correlation was found between inactivity of the tk gene and methylation of only one of the two cytosines in the EcoRI recognition site. The results also show that the tk promoter can be active despite the presence of a methylated cytosine.

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.

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.