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Influence of repair on mutations by oxidative DNA damage
Abstracts / Mutation Research 360 (1996) 201-300
jects were healthy, non-smoking males and the mutant frequency ranged between 1.39-22.5 × 10-6. Among 462 T-cell mutants studied by multiplex-PCR of genomic DNA, only 13 (2.8%) deletions were found; three total deletions, six partial exon deletions and four mutants with one or two exons deleted. Point mutations were classified in 315 mutants using reverse transcriptase - PCR amplification. 50 (15.8%) of these had splice site mutations and 254 (80.6%) had coding errors. Splice mutation was more frequent among the garage workers (18%) as compared to the controls (11.8%), possibly reflecting a PAHspecific mutation induction in these workers. Our results also shows that both gene deletion and splice mutation at the hprt locus in T-cells of healthy non-smokers could be less frequent than previously reported. The deletion breakpoints have been sequenced for five of the mutants with alterations in their multiplex-PCR pattern so far. Four mutants had at least one of their breakpoints in exon 2, one of these had a compound deletion. The fifth mutant had a 34 bp long deletion in the acceptor site of intron 3, resulting in the skipping of exon 4 in its cDNA. Non-homologous recombination seems to be the deletion mechanism in all these mutants, since no or just a few basepairs homology were found at the deletion breakpoints.
207
vated psoralens. The increased genomic instability is reflected at the gene level by a substantial increase in intragenic deletions. Indeed, in normal cells the vast majority of spontaneous and psoralen photoinduced mutations at the H P R T locus are base substitutions, whereas in FA cells the deletion mutations predominate. In order to obtain information on the mechanism(s) underlying the genomic rearrangements in FA, we examined the sequences at the breakpoints of deletions detected at the endogenous H P R T gene or at appropriate exogenous substrate. The patterns of spontaneous and psoralen photoinduced FA deletions suggest that the action of a site-specific mechanism may take place. Indeed, a significant proportion of independent deletions has a common deletion sequence at the junctions. In most of the break-sites these motifs closely matched the consensus heptamer sequence, which is the essential element of the recognition signal sequences which direct cleavage and joining in the V (D)J recombination process. Our data suggest that the mutations in the FA gene(s) may trigger an illegitimate site-specific recombination activity which will be responsible at least in part, for the deletion proneness and the chromosomal instability in this disorder. The question arises whether this activity might be an aberrant V (D) J-like activity.
1-11
1-12
An illegitimate site-specific recombination is likely to be involved in the overproduction of deletions in Fanconi anemia cells D. Papadpoulo, A. Laquerbe, N. Doyen a, S. Kallenbach ~, J.C. Andrau, F. Rougeon a, E. Moustacchi; URA 1292 du CNRS, Inst. Curie-Biologie, Paris, a URA 361 du CNRS, Inst. Pasteur, Paris Fanconi anemia (FA) belongs to a group of human inherited disorders that associate genomic instability and anomalies in the processing of specific DNA lesions to cancer proneness. FA cells show an increased level of chromosomal breakage, either spontaneous or induced by DNA cross-linking agents such as mitomycin C, diepoxybutane or photoacti-
Influence of repair on mutations by oxidative DNA damage J. Ret~l, J.E.F. Braun, A.H. Wanamarta, F. Sarquis, E.J. Westmijze, M.V.M. Lafleur; Department of Medical Oncology, Vrije Universiteit, Amsterdam, The Netherlands The mutations caused by OH radicals both under oxic ( 0 2) and anoxic (N20, N 2) conditions were investigated by ~/-irradiation of dilute aqueous solutions of ds M13mpl0 DNA, which contains the lacZa gene and a 144 basepair (bp) in frame insert as mutational target sequences. After introduction of the irradiated DNA into E. coli, the mutations formed were characterized by sequence analysis. The main
208
Abstracts/Mutation Research 360 (1996) 201-300
conclusions from these experiments were, that bp substitutions are the main mutational event ( ~ 7 0 80% of all mutations) and occur predominantly on G / C bp's; the types of bp substitution are dependent on the gassing conditions during irradiation, the local DNA conformation, the mutational target sequence and the replicon, in which the mutational target is incorporated. Frameshifts are in the minority and are mainly deletions; - 1 bp deletions are only formed by OH under anoxic (N20) conditions. The influence of repair was studied in 3 different ways: (1) Irradiation of single-stranded (ss) M13mpl0 DNA (virtually no repair). (2) Irradiation of ds DNA followed by introduction of the irradiated DNA into a nucleotide-excision-repair-deficient (uvrA-) E. coli. (3) Irradiation of dsDNA in the presence of cystamine ('chemical repair'). After irradiation of ssDNA only base substitutions were found, which occur only on G's and C's. Oxidative DNA damages on C' or G's can lead to different substitutions, the following being formed: G --* C, G --* T, C ~ G and C ~ T. In contrast, after exposure of ss M13mpl0 DNA to singlet oxygen (IO 2) more than 50% of the mutations consists of - 1 bp deletions, while the remaining bp substitutions are mainly G T transversions. The nucleotide-excision repair appeared to be involved in to the removal of DNA damage which can lead to frameshifts, in particular - 1 bp deletions, and in the site-specific removal of damage which lead to A / T ~ G / C transitions. Finally, chemical repair or modification by thiol-compounds is mainly effective on potentially lethal and not so much on mutagenic damage.
radiation protection of astronauts during long-term missions in deep space. Additionally, beams of heavy ions are going to be introduced in radiotherapy of cancers. We investigated the molecular quality of mutations in the hprt gene locus of V79 Chinese hamster cells using the Southern blot technique and hprt cDNA (plasmid pHpt 12). The damage was divided into three categories: T = total deletions, P = partial deletions, N = no visible changes, point mutations and/or small deletions. All the spontaneous mutants studied (n = 30) showed category N mutations. The X-ray-induced mutations (n = 4 0 ) showed 50-60% severe gene damage (category T + P). After irradiation with various beams of heavy ions differing in their LET-values (keV/p~m) the total of 200 mutant clones was investigated. In these cases the severity of gene damage (category T + P) increased to 100% with LET up to 500-1000 keV/p~m. In the LET range above 1000 k e V / t x m the mutation spectrum was comparable to that obtained after X-irradiation. Knowing the restriction map of the hamster hprt gene (1.4 kbp) and performing Southern blots after treatment with three different restriction enzymes (EcoRI, PstI and BglII) it was possible to localize the damage. This damage is not randomly distributed. With heavy ions and X-rays 70% and 5 0 60% of the breaks, respectively, occur between exons 4 and 7. This finding points to scaffold-attached regions between these exons, with exon 5 consisting of 18 bp only. 1-14
1-13
Analysis of hprt mutants induced by accelerated heavy ions and X-rays H. Rink, I. Rosendahl, L. Kraheck, D. Melchior; Experimental Radiology and Radiation Biology Radiology Department, University of Bonn, Bonn, Germany Highly accelerated heavy ions, being part of the cosmic radiations, are of special interest in view of
A set of Saccharomyces cerevisiae tester strains to detect carcinogen-induced translocation, gene conversion and loss of heterozygosity C. Sengstag, M. Fasullo ~, G. Acufia, F.E. Wiirgler; Institute of Toxicology, Swiss Federal Institute of Technology, Schorenstr. 16, 8603 Schwerzenbach, Switzerland, ~ Loyola University Chicago, Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA
jects were healthy, non-smoking males and the mutant frequency ranged between 1.39-22.5 × 10-6. Among 462 T-cell mutants studied by multiplex-PCR of genomic DNA, only 13 (2.8%) deletions were found; three total deletions, six partial exon deletions and four mutants with one or two exons deleted. Point mutations were classified in 315 mutants using reverse transcriptase - PCR amplification. 50 (15.8%) of these had splice site mutations and 254 (80.6%) had coding errors. Splice mutation was more frequent among the garage workers (18%) as compared to the controls (11.8%), possibly reflecting a PAHspecific mutation induction in these workers. Our results also shows that both gene deletion and splice mutation at the hprt locus in T-cells of healthy non-smokers could be less frequent than previously reported. The deletion breakpoints have been sequenced for five of the mutants with alterations in their multiplex-PCR pattern so far. Four mutants had at least one of their breakpoints in exon 2, one of these had a compound deletion. The fifth mutant had a 34 bp long deletion in the acceptor site of intron 3, resulting in the skipping of exon 4 in its cDNA. Non-homologous recombination seems to be the deletion mechanism in all these mutants, since no or just a few basepairs homology were found at the deletion breakpoints.
207
vated psoralens. The increased genomic instability is reflected at the gene level by a substantial increase in intragenic deletions. Indeed, in normal cells the vast majority of spontaneous and psoralen photoinduced mutations at the H P R T locus are base substitutions, whereas in FA cells the deletion mutations predominate. In order to obtain information on the mechanism(s) underlying the genomic rearrangements in FA, we examined the sequences at the breakpoints of deletions detected at the endogenous H P R T gene or at appropriate exogenous substrate. The patterns of spontaneous and psoralen photoinduced FA deletions suggest that the action of a site-specific mechanism may take place. Indeed, a significant proportion of independent deletions has a common deletion sequence at the junctions. In most of the break-sites these motifs closely matched the consensus heptamer sequence, which is the essential element of the recognition signal sequences which direct cleavage and joining in the V (D)J recombination process. Our data suggest that the mutations in the FA gene(s) may trigger an illegitimate site-specific recombination activity which will be responsible at least in part, for the deletion proneness and the chromosomal instability in this disorder. The question arises whether this activity might be an aberrant V (D) J-like activity.
1-11
1-12
An illegitimate site-specific recombination is likely to be involved in the overproduction of deletions in Fanconi anemia cells D. Papadpoulo, A. Laquerbe, N. Doyen a, S. Kallenbach ~, J.C. Andrau, F. Rougeon a, E. Moustacchi; URA 1292 du CNRS, Inst. Curie-Biologie, Paris, a URA 361 du CNRS, Inst. Pasteur, Paris Fanconi anemia (FA) belongs to a group of human inherited disorders that associate genomic instability and anomalies in the processing of specific DNA lesions to cancer proneness. FA cells show an increased level of chromosomal breakage, either spontaneous or induced by DNA cross-linking agents such as mitomycin C, diepoxybutane or photoacti-
Influence of repair on mutations by oxidative DNA damage J. Ret~l, J.E.F. Braun, A.H. Wanamarta, F. Sarquis, E.J. Westmijze, M.V.M. Lafleur; Department of Medical Oncology, Vrije Universiteit, Amsterdam, The Netherlands The mutations caused by OH radicals both under oxic ( 0 2) and anoxic (N20, N 2) conditions were investigated by ~/-irradiation of dilute aqueous solutions of ds M13mpl0 DNA, which contains the lacZa gene and a 144 basepair (bp) in frame insert as mutational target sequences. After introduction of the irradiated DNA into E. coli, the mutations formed were characterized by sequence analysis. The main
208
Abstracts/Mutation Research 360 (1996) 201-300
conclusions from these experiments were, that bp substitutions are the main mutational event ( ~ 7 0 80% of all mutations) and occur predominantly on G / C bp's; the types of bp substitution are dependent on the gassing conditions during irradiation, the local DNA conformation, the mutational target sequence and the replicon, in which the mutational target is incorporated. Frameshifts are in the minority and are mainly deletions; - 1 bp deletions are only formed by OH under anoxic (N20) conditions. The influence of repair was studied in 3 different ways: (1) Irradiation of single-stranded (ss) M13mpl0 DNA (virtually no repair). (2) Irradiation of ds DNA followed by introduction of the irradiated DNA into a nucleotide-excision-repair-deficient (uvrA-) E. coli. (3) Irradiation of dsDNA in the presence of cystamine ('chemical repair'). After irradiation of ssDNA only base substitutions were found, which occur only on G's and C's. Oxidative DNA damages on C' or G's can lead to different substitutions, the following being formed: G --* C, G --* T, C ~ G and C ~ T. In contrast, after exposure of ss M13mpl0 DNA to singlet oxygen (IO 2) more than 50% of the mutations consists of - 1 bp deletions, while the remaining bp substitutions are mainly G T transversions. The nucleotide-excision repair appeared to be involved in to the removal of DNA damage which can lead to frameshifts, in particular - 1 bp deletions, and in the site-specific removal of damage which lead to A / T ~ G / C transitions. Finally, chemical repair or modification by thiol-compounds is mainly effective on potentially lethal and not so much on mutagenic damage.
radiation protection of astronauts during long-term missions in deep space. Additionally, beams of heavy ions are going to be introduced in radiotherapy of cancers. We investigated the molecular quality of mutations in the hprt gene locus of V79 Chinese hamster cells using the Southern blot technique and hprt cDNA (plasmid pHpt 12). The damage was divided into three categories: T = total deletions, P = partial deletions, N = no visible changes, point mutations and/or small deletions. All the spontaneous mutants studied (n = 30) showed category N mutations. The X-ray-induced mutations (n = 4 0 ) showed 50-60% severe gene damage (category T + P). After irradiation with various beams of heavy ions differing in their LET-values (keV/p~m) the total of 200 mutant clones was investigated. In these cases the severity of gene damage (category T + P) increased to 100% with LET up to 500-1000 keV/p~m. In the LET range above 1000 k e V / t x m the mutation spectrum was comparable to that obtained after X-irradiation. Knowing the restriction map of the hamster hprt gene (1.4 kbp) and performing Southern blots after treatment with three different restriction enzymes (EcoRI, PstI and BglII) it was possible to localize the damage. This damage is not randomly distributed. With heavy ions and X-rays 70% and 5 0 60% of the breaks, respectively, occur between exons 4 and 7. This finding points to scaffold-attached regions between these exons, with exon 5 consisting of 18 bp only. 1-14
1-13
Analysis of hprt mutants induced by accelerated heavy ions and X-rays H. Rink, I. Rosendahl, L. Kraheck, D. Melchior; Experimental Radiology and Radiation Biology Radiology Department, University of Bonn, Bonn, Germany Highly accelerated heavy ions, being part of the cosmic radiations, are of special interest in view of
A set of Saccharomyces cerevisiae tester strains to detect carcinogen-induced translocation, gene conversion and loss of heterozygosity C. Sengstag, M. Fasullo ~, G. Acufia, F.E. Wiirgler; Institute of Toxicology, Swiss Federal Institute of Technology, Schorenstr. 16, 8603 Schwerzenbach, Switzerland, ~ Loyola University Chicago, Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA