Artemis splice defects cause atypical SCID and can be restored in vitro by an antisense oligonucleotide.

Artemis deficiency is known to result in classical T-B- severe combined immunodeficiency (SCID) in case of Artemis null mutations, or Omenn's syndrome in case of hypomorphic mutations in the Artemis gene. We describe two unrelated patients with a relatively mild clinical T-B- SCID phenotype, caused by different homozygous Artemis splice-site mutations. The splice-site mutations concern either dysfunction of a 5' splice-site or an intronic point mutation creating a novel 3' splice-site, resulting in mutated Artemis protein with residual activity or low levels of wild type (WT) Artemis transcripts. During the first 10 years of life, the patients suffered from recurrent infections necessitating antibiotic prophylaxis and intravenous immunoglobulins. Both mutations resulted in increased ionizing radiation sensitivity and insufficient variable, diversity and joining (V(D)J) recombination, causing B-lymphopenia and exhaustion of the naive T-cell compartment. The patient with the novel 3' splice-site had progressive granulomatous skin lesions, which disappeared after stem cell transplantation (SCT). We showed that an alternative approach to SCT can, in principle, be used in this case; an antisense oligonucleotide (AON) covering the intronic mutation restored WT Artemis transcript levels and non-homologous end-joining pathway activity in the patient fibroblasts.

Human and mouse homologs of the Saccharomyces cerevisiae RAD54 DNA repair gene: evidence for functional conservation.

BACKGROUND: Homologous recombination is of eminent importance both in germ cells, to generate genetic diversity during meiosis, and in somatic cells, to safeguard DNA from genotoxic damage. The genetically well-defined RAD52 pathway is required for these processes in the yeast Saccharomyces cerevisiae. Genes similar to those in the RAD52 group have been identified in mammals. It is not known whether this conservation of primary sequence extends to conservation of function. RESULTS: Here we report the isolation of cDNAs encoding a human and a mouse homolog of RAD54. The human (hHR54) and mouse (mHR54) proteins were 48% identical to Rad54 and belonged to the SNF2/SW12 family, which is characterized by amino-acid motifs found in DNA-dependent ATPases. The hHR54 gene was mapped to chromosome 1p32, and the hHR54 protein was located in the nucleus. We found that the levels of hHR54 mRNA increased in late G1 phase, as has been found for RAD54 mRNA. The level of mHR54 mRNA was elevated in organs of germ cell and lymphoid development and increased mHR54 expression correlated with the meiotic phase of spermatogenesis. The hHR54 cDNA could partially complement the methyl methanesulfonate-sensitive phenotype of S. cerevisiae rad54 delta cells. CONCLUSIONS: The tissue-specific expression of mHR54 is consistent with a role for the gene in recombination. The complementation experiments show that the DNA repair function of Rad54 is conserved from yeast to humans. Our findings underscore the fundamental importance of DNA repair pathways: even though they are complex and involve multiple proteins, they seem to be functionally conserved throughout the eukaryotic kingdom.

The Drosophila melanogaster DmRAD54 gene plays a crucial role in double-strand break repair after P-element excision and acts synergistically with Ku70 in the repair of X-ray damage.

The RAD54 gene has an essential role in the repair of double-strand breaks (DSBs) via homologous recombination in yeast as well as in higher eukaryotes. A Drosophila melanogaster strain deficient in the RAD54 homolog DmRAD54 is characterized by increased X-ray and methyl methanesulfonate (MMS) sensitivity. In addition, DmRAD54 is involved in the repair of DNA interstrand cross-links, as is shown here. However, whereas X-ray-induced loss-of-heterozygosity (LOH) events were completely absent in DmRAD54(-/-) flies, treatment with cross-linking agents or MMS resulted in only a slight reduction in LOH events in comparison with those in wild-type flies. To investigate the relative contributions of recombinational repair and nonhomologous end joining in DSB repair, a DmRad54(-/-)/DmKu70(-/-) double mutant was generated. Compared with both single mutants, a strong synergistic increase in X-ray sensitivity was observed in the double mutant. No similar increase in sensitivity was seen after treatment with MMS. Apparently, the two DSB repair pathways overlap much less in the repair of MMS-induced lesions than in that of X-ray-induced lesions. Excision of P transposable elements in Drosophila involves the formation of site-specific DSBs. In the absence of the DmRAD54 gene product, no male flies could be recovered after the excision of a single P element and the survival of females was reduced to 10% compared to that of wild-type flies. P-element excision involves the formation of two DSBs which have identical 3' overhangs of 17 nucleotides. The crucial role of homologous recombination in the repair of these DSBs may be related to the very specific nature of the breaks.

The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination.

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRADS4-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution.

Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation.

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52(-/-) ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.

Molecular analysis of mutations induced in the vermilion gene of Drosophila melanogaster by methyl methanesulfonate.

The nature of DNA sequence changes induced by methyl methanesulfonate (MMS) at the vermilion locus of Drosophila melanogaster was determined after exposure of postmeiotic male germ cell stages. MMS is a carcinogen with strong preference for base nitrogen alkylation (s = 0.86). The spectrum of 40 intralocus mutations was dominated by AT----GC transitions (23%), AT----TA transversions (54%) and deletions (14%). The small deletions were preferentially found among mutants isolated in the F1 (8/18), whereas the AT----GC transitions exclusively occurred in the F2 (6/22). The MMS-induced transversions and deletions are presumably caused by N-methyl DNA adducts, which may release apurinic intermediates, known to be a time-related process. Furthermore, MMS produces multilocus deletions, i.e., at least 30% of the F1 mutants analyzed were of this type. A comparison of the mutational spectra of MMS with that produced by ethylnitrosourea (ENU), also in the vermilion locus of Drosophila, reveals major differences: predominantly transition mutations (61% GC----AT and 18% AT----GC) were found in both the F1 and F2 spectrum induced by ENU. It is concluded that the mutational spectrum of MMS is dominated by nitrogen DNA adducts, whereas with ENU DNA sequence changes mainly arose from modified oxygen in DNA.

Sequence analysis of N-ethyl-N-nitrosourea-induced vermilion mutations in Drosophila melanogaster.

The mutational specificity of N-ethyl-N-nitrosourea (ENU) was determined in Drosophila melanogaster using the vermilion locus as a target gene. 25 mutants (16 F1 and 9 F2 mutants) were cloned and sequenced. Only base-pair changes were observed; three of the mutants represented double base substitutions. Transition mutations were the most prominent sequence change: 61% were GC----AT and 18% AT----GC substitutions. Both sequence changes can be explained by the miscoding properties of the modified guanine and thymine bases. A strong bias of neighboring bases on the occurrence of the GC----AT transitions or a strand preference of both types of transition mutations was not observed. The spectrum of ENU mutations in D. melanogaster includes a significant fraction (21%) of transversion mutations. Our data indicate that like in other prokaryotic and eukaryotic systems also in D. melanogaster the O6-ethylguanine adduct is the most prominent premutational lesion after ENU treatment. The strong contribution of the O6-ethylguanine adduct to the mutagenicity of ENU possibly explains the absence of distinct difference between the type of mutations observed in the F1 and F2 mutants. Although the latter arise later during development, the spectrum of mosaic mutations is also dominated by GC----AT transition mutations.

The nature of N-ethyl-N-nitrosourea-induced mutations at the white locus of Drosophila melanogaster.

The molecular basis of 36 mutations induced by N-ethyl-N-nitrosourea (ENU) at the white locus of Drosophila melanogaster was analyzed. Blot-hybridization showed that only two of them are rearrangements. One is a 200-bp deletion and the second mutant is an insertion of about 10 kb. The latter might be of spontaneous origin. 34 mutants did not show a detectable alteration of the normal restriction enzyme profile. 21 mutants were also analyzed by Northern blot-hybridization. Normal or nearly normal levels of white mRNA were observed in 8 pigmented and 7 non-pigmented mutants. In 5 other non-pigmented mutants a strong reduction of the amount of mature white mRNA was seen. In one of the pigmented mutants, hybridization occurred with 2 RNAs. When taken together, these results strongly indicate that most of the ENU-induced mutations are caused by base-pair changes or rearrangements smaller than 50-100 bp.

Genomic integrity and the repair of double-strand DNA breaks.

The induction of double-strand breaks (DSBs) in DNA by exposure to DNA damaging agents or as intermediates in normal cellular processes, creates a severe threat for the integrity of the genome. Unrepaired or incorrectly repaired DSBs lead to broken chromosomes and/or gross chromosomal rearrangements which are frequently associated with tumor formation in mammals. To maintain the integrity of the genome and to prevent the formation of chromosomal aberrations, several pathways exist in eukaryotes: homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). These mechanisms are conserved in evolution, but the relative contribution depends on the organism, cell type and stage of the cell cycle. In yeast, DSBs are primarily repaired via HR while in higher eukaryotes, both HR and NHEJ are important. In mammals, defects in both HR or NHEJ lead to a predisposition to cancer and at the cellular level, the frequency of chromosomal aberrations is increased. This review summarizes our current knowledge about DSB-repair with emphasis on recent progress in understanding the precise biochemical activities of individual proteins involved.

Genomic characterization of the mouse homolog of the Saccharomyces cerevisiae recombination and double-strand break repair gene RAD52.

The yeast Saccharomyces cerevisiae RAD52 gene is involved in recombination and DNA double-strand break repair. Recently, mouse and human homologs of the yeast RAD52 gene have been identified. Here we present the genomic organization of the mouse RAD52 gene. It consists of 12 exons ranging in size from 67 to 374 bp spread over a region of approximately 18 kb. The first ATG is located in exon 2. Analysis of the promoter region revealed no classical promoter elements such as CCAAT or TATA boxes. Transcriptional mapping analysis revealed one major transcription start point. Analogous to the situation in yeast, transcription of the RAD52 gene in human skin fibroblasts and mouse Ltk- cells was not induced by methyl methanesulfonate treatment. Furthermore, no specific alteration in human RAD52 expression levels throughout the cell cycle was observed.

Mutational spectra induced under distinct excision repair conditions by the 3 methylating agents N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine and N-nitrosodimethylamine in postmeiotic male germ cells of Drosophila.

This paper describes the analysis of mutations induced at the vermilion locus in postmeiotic male germ cell stages of Drosophila exposed to 3 different N-methyl-N-nitroso compounds: N-methyl-N-nitrosourea (MNU); N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); and N-nitrosodimethylamine (DMN). With MNU and DMN, the impact of DNA nucleotide excision repair (NER) on the spectra of mutations was studied. Mutants were isolated from F1 (mutations fixed before the first mitotic replication after fertilization) and F2 (mutations fixed following one or more mitotic replications; mosaics in F1) generations. The vermilion system enables the analysis of both intra- and inter-locus DNA changes for which several techniques have been adapted: (1) amplification of the vermilion gene by PCR, cloning of the fragment and sequence analysis of ssDNA; (2) Southern blot hybridization; and (3) cytological analysis of polytene chromosomes. In total, 49 MNU (26 from the exr+ genotype and 23 from the exr- genotype), 47 DMN (28 from the exr+ genotype and 19 from the exr- genotype) and 16 MNNG-induced mutations were characterized. The F1 spectra of all 3 agents contained base-pair changes and deletions (intra- and multi-locus) in a ratio of roughly 1 to 1, indicating a significant contribution of nitrogen DNA adducts to the spectra. In all F2 spectra the levels of base-pair changes were significantly higher compared to those in the F1 spectra, a finding also made for methyl methanesulfonate-induced mutations in earlier studies. There is an increase of mutations of, especially, the transversion types of mutations under exr- conditions in comparison to the exr+ situation. The induced transversions, clearly present in all spectra (exr+ and exr-), are presumably caused by N-methyl DNA adducts, which upon release from the DNA backbone lead to apurinic sites in a time-related process. Regarding the occurrence of transitions, it turned out for all 3 mutagens that the AT-->GC type strongly dominated the GC-->AT transitions. This suggest that O6-methylguanine is efficiently repaired, in contrast to O4-methylthymine. Based on the data obtained in the vermilion system with ENU, we propose, in addition, that the Drosophila alkyltransferase system repairs O6-methylguanine more efficiently than O6-ethylguanine.

Mutations induced at the white and vermilion loci in Drosophila melanogaster.

The white and vermilion loci in D. melanogaster were selected as target genes for the study of the mutational specificity of ionizing radiation and N-ethyl-N-nitrosourea (ENU) in a whole organism. Analysis of X-ray- and neutron-induced white mutants by a combination of genetic and molecular techniques showed that ionizing radiation induces primarily break-type mutations against a repair-proficient background, the majority of these alterations being deletions. Both very large multi-locus deficiencies and deletions of only a few base pairs were observed. These small deletions are flanked by repeats of 2-3 nucleotides, one copy of which is retained at the new junction. Presumably these small repeats are involved in the generation of the X-ray-induced deletions. In excision-repair-deficient mus201D1 flies, the frequency of whole-body white mutants recovered after X-ray irradiation is the same as in the wild-type strain. The percentage of mosaic mutations, however, is enhanced by a factor 3-4. Analysis by blot hybridization of ENU-induced white mutants strongly indicates that most mutations are due to base-pair changes. This was confirmed by sequence analysis of 25 ENU-induced vermilion mutants. In all mutants the alterations are due to base-pair changes, the majority being GC to AT transitions (61%).

DNA sequence analysis of X-ray-induced deletions at the white locus of Drosophila melanogaster.

We have determined the nucleotide sequence of 5 X-ray-induced white mutants containing small rearrangements. Comparison with wild-type sequences showed deletions in the coding region ranging in size between 6 bp and 29 bp. These small deletions are distributed non-randomly over the white locus. Two mutants contain the same 29-bp deletion, while the other 3 deletions are clustered. All 5 deletions have occurred between 2 and 3 bp repeats. One of the repeats is preserved in the novel junction formed by the deletion. Our results suggest that recombinational processes may be involved in the generation of X-ray-induced deletions.

The nature of X-ray-induced mutations after recovery in excision repair-deficient (mus-201) Drosophila females.

This paper describes the genetic analysis of X-ray-induced mutations at several visible loci (yellow, white, Notch, vermilion and forked) located on the X-chromosome of Drosophila melanogaster after recovery in excision repair-deficient condition (mus-201). A total of 118 mutations observed in 83636 F1 females were analyzed. The white mutations in particular have been investigated at the molecular level. The results show that: (1) the frequency of recovered whole-body mutations is similar or slightly lower in repair-deficient than in repair-proficient condition (respectively 1.5 x 10(-4)/locus/15 Gy and 2.3 x 10(-4)/locus/15 Gy); (2) the frequency of observed mosaic mutations is significantly higher in the repair-deficient condition than in the proficient condition (respectively 2.7 x 10(-4)/locus/15 Gy and 0.9 x 10(-4)/locus/15 Gy); (3) the analysis of F2 male lethal mutations and the cytological analysis of the recovered mutations in the excision repair-deficient condition indicate a decrease in mutations associated with gross chromosomal aberrations (including multilocus deletions); (4) at the molecular level, the spectrum of recovered intragenic mutations is similar after excision-deficient and -proficient repair. These results indicate that excision repair is involved in X-ray-induced DNA damage that is repaired efficiently in the normal repair condition, but bypassed in the excision repair-deficient condition, leading to mosaic mutations. In addition, lesions that apparently cannot be bypassed by DNA replication lead to a decrease in the fraction of mutations due to gross chromosomal aberrations among the whole-body mutations.

The nature of radiation-induced mutations at the white locus of Drosophila melanogaster.

X-Ray- and neutron-induced mutations at the white locus of Drosophila melanogaster were used to study the nature of radiation-induced genetic damage. Genetic analysis showed the presence of multi-locus deficiencies in 15 out of 31 X-ray mutants and in 26 out of 35 mutants induced by neutrons. The DNA from 11 X-ray and 4 neutron mutants, which were not multi-locus deficiencies, was analyzed by Southern blot-hybridization. Deletions were observed in 2 X-ray and 1 neutron mutant. In combination with cytogenetic techniques, chromosomal rearrangements affecting the white locus (translocations, inversions, etc.) were identified in 3 X-ray and in 2 neutron mutants. A hot-spot for translocation breakpoints was identified in the left arm of the third chromosome. 5 X-ray mutants, which apparently did not contain large deletions, were subjected to further analysis by the nuclease S1 protection method, after cloning of the white gene. In 4 mutants a small deletion could indeed be detected in this way. Thus it seems that by far the main part of X-ray- and neutron-induced white mutants have arisen through large changes in the white gene, especially deletions.

Isolation and genetic characterisation of the Drosophila homologue of (SCE)REV3, encoding the catalytic subunit of DNA polymerase zeta.

In Drosophila, about 30 mutants are known that show hypersensitivity to the methylating agent methyl methane sulfonate (MMS). Addition of this agent to the medium results in an increased larval mortality of the mutants. Using a P-insertion mutagenesis screen, three MMS-sensitive mutants on chromosome II were isolated. One of these is allelic to the known EMS-induced mus205 (mutagen sensitive) mutant. In the newly isolated mutant, a P-element is detected in region 43E by in situ hybridisation. The localisation of mus205 to this region was confirmed by deficiency mapping. The gene was cloned and shows strong homology to the Saccharomyces cerevisiae REV3 gene. The REV3 gene encodes the catalytic subunit of DNA polymerase zeta, involved in translesion synthesis. The P-element is inserted in the first exon of the mus205 gene resulting in an aberrant mRNA, encoding a putative truncated protein containing only the first 13 of the 2130 aa native Drosophila protein. The mus205 mutant is hypersensitive to alkylating agents and UV, but not to ionising radiation. In contrast to reported data, in germ cells, the mutant has no effect on mutability by X-rays, NQO and alkylating agents. In somatic cells, the mutant shows no effect on MMS-induced mutations and recombinations. This phenotype of the Drosophila mus205 mutant is strikingly different from the phenotype of the yeast rev3 mutant, which is hypomutable after UV, X-rays, NQO and alkylating agents.

Evaluation of the database on mutant frequencies and DNA sequence alterations of vermilion mutations induced in germ cells of Drosophila shows the importance of a neutral mutation detection system.

The vermilion gene in Drosophila has extensively been used for the molecular analysis of mutations induced by chemicals in germ cells in vivo. The gene is located on the X-chromosome and is a useful target for the study of mutagenesis since all types of mutations are generated. We have critically evaluated this system with respect to sensitivity for mutation induction and selectivity for different types of mutations, using a database of more than 600 vermilion mutants induced in postmeiotic male germ cells by 18 mutagens. From most of these mutants the mutation has been analysed. These data showed 336 base substitutions, 96 intra-locus DNA rearrangements and 78 multi-locus deletions (MLD). Mutants containing a MLD were either heterozygous sterile or homozygous and hemizygous lethal. The distribution of both basepair (bp) changes and intra-locus rearrangements over the coding region of the vermilion gene was uniform with no preferences concerning 5' or 3' regions, certain exons, splice sites, specific amino acid changes or nonsense mutations. Possible hotspots for base substitutions seem to be related to the type of DNA damage rather than to the vermilion system. Gene mutations other than bp changes were examined on sequence characteristics flanking the deletion breakpoints. Induction frequencies of vermilion mosaic mutants were, in general, higher than those of vermilion complete mutants, suggesting that persistent lesions are the main contributors to the molecular spectra. Comparison of induction frequencies of vermilion mutants and sex-linked recessive lethal (SLRL) mutants for the 18 mutagens showed that the sensitivity of the vermilion gene against a mutagenic insult is representative for genes located on the X-chromosome. The effect of nucleotide excision repair (NER) on the formation of SLRL mutants correlated with an increase of transversions in the vermilion spectra under NER deficient conditions. Furthermore, the clastogenic potency of the mutagens, i.e., the efficiency to induce chromosomal-losses vs. SLRL forward mutations, shows a positive correlation with the percentage of DNA deletions in the molecular spectra of vermilion mutants.

Characterization of RAD52 homologs in the fission yeast Schizosaccharomyces pombe.

The RAD52 gene of Saccharomyces cerevisiae is essential for repair of DNA double-strand breaks (DSBs) by homologous recombination. Inactivation of this gene confers hypersensitivity to DSB-inducing agents and defects in most forms of recombination. The rad22+ gene in Schizosaccharomyces pombe (here referred to as rad22A+) has been characterized as a homolog of RAD52 in fission yeast. Here, we report the identification of a second RAD52 homolog in Schizosaccharomyces pombe, called rad22B+. The amino acid sequences of Rad22A and Rad22B show significant conservation (38% identity). Deletion mutants of respectively, rad22A and rad22B, show different phenotypes with respect to sensitivity to X-rays and the ability to perform homologous recombination as measured by the integration of plasmid DNA. Inactivation of rad22A+ leads to a severe sensitivity to X-rays and a strong decrease in recombination (13-fold), while the rad22B mutation does not result in a decrease in homologous recombination or a change in radiation sensitivity. In a rad22A-rad22B double mutant the radiation sensitivity is further enhanced in comparison with the rad22A single mutant. Overexpression of the rad22B+ gene results in partial suppression of the DNA repair defects of the rad22A mutant strain. Meiotic recombination and spore viability are only slightly affected in either single mutant, but outgrowth of viable spores is almost 31-fold reduced in the rad22A-rad22B double mutant. The results obtained imply a crucial role for rad22A+ in repair and recombination in vegetative cells just like RAD52 in S. cerevisiae. The rad22B+ gene presumably has an auxiliary role in the repair of DSBs. The drastic reduced spore viability in the double mutant suggests that meiosis in S. pombe is dependent on the presence of either rad22A+ or rad22B+.

The nature of X-ray-induced mutations in mature sperm and spermatogonial cells of Drosophila melanogaster.

Mutations at four X-linked visible loci (yellow, white, vermilion and forked) induced by X-irradiation of mature sperm and spermatogonial cells were analysed genetically and cytogenetically. In addition, a fraction of the intragenic vermilion mutations was analysed molecularly. Males of two wild-type strains (Amherst M56i and Berlin-K) were used. A total of 332,651 chromosomes of irradiated mature sperm and 311,567 of irradiated spermatogonial cells were scored. The ratio of F1 female sterile, F2 male lethal and F2 male viable mutations in mature sperm and spermatogonial cells is very similar. The cytogenetic analysis shows equal fractions of multilocus deletions and translocations among the mutations recovered from both stages of spermatogenesis. These data strongly suggest that the spectrum of X-ray mutations is similar in mature sperm and spermatogonial cells, including multilocus deletions and chromosome rearrangements. The molecular analysis of a number of intragenic vermilion mutations showed the presence of three small deletions (1-10 bp), one insertion of two nucleotides and seven single nucleotide changes.

The nature of X-ray and chemically induced mutations in Drosophila in relation with DNA repair.

This paper describes the spectrum of mutations induced by alkylating agents and ionizing radiation in Drosophila. Specifically, the genotoxic profile of the alkylating agents is set against their carcinogenic potency. Alkylating agents that react preferentially with N-atoms in the DNA are relatively poor mutagens, especially so in repair-competent (early) germ cells, and likewise weak carcinogens when compared to those that are more efficient in O-alkylation. Genetic techniques combined with molecular analysis of X-ray and neutron induced mutations show that ionizing radiation induces primarily break-type mutations in a repair proficient background. Both multi-locus deletions as well as small intragenic deletions of only a few base-pairs are observed. The small deletions occur between direct repeats of 2-3 nucleotides, one copy of which is retained in the mutant allele. Possibly, these deletions are the result of repair processes. The effect of changes in DNA-repair (excision repair deficient) is reflected by a "hypermutability" for alkylating agents specifically for N-alkylators, indicating that the normal efficient error-free repair of N-alkylation damage can explain the high exposure doses required for tumor induction in mammals. The frequency of X-ray induced whole-body white mutations, recovered in excision repair deficient Drosophila, is only slightly enhanced, when compared to the repair proficient situation. In contrast, mosaic mutations occur 3-4 times more frequent, indicating that part of the X-ray damage, normally removed by the excision repair process, is not a major impedement during replication.(ABSTRACT TRUNCATED AT 250 WORDS)