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.

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.

Isolation and characterization of the RAD59 homologue of Kluyveromyces lactis.

Homologous recombination in the yeast Saccharomyces cerevisiae is under the control of the RAD52 epistasis group. Genes belonging to this group show strong conservation during evolution and homologues of most members have been identified in other eukaryotic organisms such as Schizosaccharomyces pombe, Drosophila and mammals. A homologue of the ScRAD59 gene, which shows structural and functional overlap with ScRAD52, has not been identified in other organisms until now. Previous assessment of the ScRAD59 function revealed that the product of this gene is required for certain types of ScRAD51-independent recombination and single-strand annealing. Also, in the distantly related fission yeast, Sch. pombe, a second RAD52 homologue has been identified (rad/22B+), but this gene more closely resembles ScRAD52 than ScRAD59 at the amino-acid level. In this study, the isolation of a homologue of ScRAD59 in Kluyveromyces lactis, KlRAD59, is described. A Klrad159 null allele results in moderate sensitivity to X-rays, indicating that the KlRAD59 gene is involved in the repair of X-ray-induced DNA damage. The amino acids in the putative K1Rad59 protein share 53% identity and 11% similarity with ScRad59. The KlRAD59 gene fully complements both the X-ray-sensitive phenotype and defects in recombination of the Scrad59 mutant strain. Our results underscore the evolutionary conservation of the RAD52 group of genes and provide evidence that the presence of additional RAD52 homologues is not limited to Sac. cerevisiae and Sch. pombe and might be a general phenomenon.

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.

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+.

Analyses of TCRB rearrangements substantiate a profound deficit in recombination signal sequence joining in SCID foals: implications for the role of DNA-dependent protein kinase in V(D)J recombination.

We reported previously that the genetic SCID disease observed in Arabian foals is explained by a defect in V(D)J recombination that profoundly affects both coding and signal end joining. As in C.B-17 SCID mice, the molecular defect in SCID foals is in the catalytic subunit of the DNA-dependent protein kinase (DNA-PKCS); however, in SCID mice, signal end resolution remains relatively intact. Moreover, recent reports indicate that mice that completely lack DNA-PKCS also generate signal joints at levels that are indistinguishable from those observed in C.B-17 SCID mice, eliminating the possibility that a partially active version of DNA-PKCS facilitates signal end resolution in SCID mice. We have analyzed TCRB rearrangements and find that signal joints are reduced by approximately 4 logs in equine SCID thymocytes as compared with normal horse thymocytes. A potential explanation for the differences between SCID mice and foals is that the mutant DNA-PKCS allele in SCID foals inhibits signal end resolution. We tested this hypothesis using DNA-PKCS expression vectors; in sum, we find no evidence of a dominant-negative effect by the mutant protein. These and other recent data are consistent with an emerging consensus: that in normal cells, DNA-PKCS participates in both coding and signal end resolution, but in the absence of DNA-PKCS an undefined end joining pathway (which is variably expressed in different species and cell types) can facilitate imperfect signal and coding end joining.

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.

Identification and characterisation of the Drosophila melanogaster O6-alkylguanine-DNA alkyltransferase cDNA.

The protein O 6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved in the repair of O 6-alkylguanine and O 4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents. A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene ( DmAGT ) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man. The DmAGT gene is located at position 84A on the third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA. Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O 6-benzylguanine. In an E.coli lac Z reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-->A:T as well as A:T-->G:C transition mutations in vivo. These results demonstrate the presence of an alkyltransferase specific for the repair of O 6-methylguanine and O 4-methylthymine in Drosophila.

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.

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.

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.

Homologous recombination in the fission yeast Schizosaccharomyces pombe: different requirements for the rhp51+, rhp54+ and rad22+ genes.

The Schizosaccharomyces pombe rhp51+, rad22+ and rhp54+ genes are homologous to RAD51, RAD52 and RAD54 respectively, which are indispensable in the recombinational repair of double-strand breaks (DSBs) in Saccharomyces cerevisiae. The rhp51Delta and rhp54Delta strains are extremely sensitive to ionizing radiation; the rad22Delta mutant turned out to be much less sensitive. Homologous recombination in these mutants was studied by targeted integration at the leu1-32 locus. These experiments revealed that rhp51Delta and rhp54Delta are equally impaired in the integration of plasmid molecules (15-fold reduction), while integration in the rad22Delta mutant is only reduced by a factor of two. Blot-analysis demonstrated that the majority of the leu+ transformants of the wild-type and rad22Delta strains have integrated one or more copies of the vector. Gene conversion events were observed in less than 10% of the transformants. Interestingly, the relative contribution of gene conversion events is much higher in a rhp51Delta and a rhp54Delta background. Meiotic recombination is hardly affected in the rad22Delta mutant. The rhp51Delta and rhp54Delta strains also show minor deficiencies in this type of recombination. The viability of spores is 46% in the rad22Delta strain and 27% in the rhp54Delta strain, as compared with wild-type cells. However, in the rhp51Delta mutant the spore viability is only 1.7%, suggesting an essential role for Rhp51 in meiosis. The function of Rhp51 and Rhp54 in damage repair and recombination resembles the role of Rad51 and Rad54 in S. cerevisiae. Compared with Rad52 from S. cerevisiae, Rad22 has a much less prominent role in the recombinational repair pathway in S. pombe.

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.

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.

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.

Isolation of the Schizosaccharomyces pombe RAD54 homologue, rhp54+, a gene involved in the repair of radiation damage and replication fidelity.

The RAD54 gene of Saccharomyces cerevisiae encodes a putative helicase, which is involved in the recombinational repair of DNA damage. The RAD54 homologue of the fission yeast Schizosaccharomyces pombe, rhp54+, was isolated by using the RAD54 gene as a heterologous probe. The gene is predicted to encode a protein of 852 amino acids. The overall homology between the mutual proteins of the two species is 67% with 51% identical amino acids and 16% similar amino acids. A rhp54 deletion mutant is very sensitive to both ionizing radiation and UV. Fluorescence microscopy of the rhp54 mutant cells revealed that a large portion of the cells are elongated and occasionally contain aberrant nuclei. In addition, FACS analysis showed an increased DNA content in comparison with wild-type cells. Through a minichromosome-loss assay it was shown that the rhp54 deletion mutant has a very high level of chromosome loss. Furthermore, the rhp54 mutation in either a rad17 or a cdc2.3w mutant background (where the S-phase/mitosis checkpoint is absent) shows a significant reduction in viability. It is hypothesized that the rhp54+ gene is involved in the recombinational repair of UV and X-ray damage and plays a role in the processing of replication-specific lesions.

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.

Cloning the RAD51 homologue of Schizosaccharomyces pombe.

The RAD51 gene of Saccharomyces cerevisiae encodes a RecA like protein, which is involved in the recombinational repair of double strand breaks. We have isolated the RAD51 homologue, rhp51+, of the distantly related yeast strain Schizosaccharomyces pombe by heterologous hybridization. DNA sequence analysis of the rhp51+ gene revealed an open reading frame of 365 amino acids. Comparison of the amino acid sequences of RAD51 and rhp51+ showed a high level of conservation: 69% identical amino acids. There are two Mlul sites in the upstream region which may be associated with cell cycle regulation of the rhp51+ gene. The rhp51+ null allele, constructed by disruption of the coding region, is extremely sensitive to X-rays, indicating that the rhp51+ gene, like RAD51, is also involved in the repair of X-ray damage. The structural and functional homology between rhp51+ and RAD51 suggests evolutionary conservation of certain steps in the recombinational repair pathway.

Impact of DNA nucleotide excision repair on methyl methanesulfonate-induced mutations in Drosophila melanogaster.

To study the impact of DNA nucleotide excision repair (NER) on the spectrum of mutations induced by alkylating agents, postmeiotic male germ cell stages of Drosophila melanogaster were exposed to methyl methanesulfonate (MMS) and the males then mated with nucleotide excision repair deficient (exr-; mus(2)201) females. MMS (s value = 0.86) has a strong preference for alkylating the nitrogen positions in DNA, whereas < 1% of all DNA lesions are on oxygen. For genetic and molecular analysis of the types of mutations induced by MMS the vermilion locus was used as target gene. Mutation induction by MMS was increased 10-fold under the exr- conditions compared to a normal functioning repair system. The genetic analysis showed that < 15% of the mutants represented inter-locus mutations, which were classified as multi-locus deletions. Of the intra-locus mutations (18 F1 and 8 F2 mutants) 78% were transversions with a clear dominance of AT-->TA (11 in the F1, 3 in the F2) and few GC-->TA (2 in the F1, 3 in the F2) type of transversions. In comparison to the MMS spectrum produced under repair proficient (exr+) condition (Nivard, M.J.M., Pastink, A. and Vogel, E.W., 1992), the exr- spectrum shows a significant decrease in the percentage of deletions and a relative increase in transversions. These data are consistent with previously published papers suggesting that under normal repair conditions the nitrogen DNA adducts are efficiently repaired in Drosophila and that the hypermutability of MMS in the exr- strain is caused by an increased formation of apurinic sites either formed from 3-methyladenine or 7-methylguanine. This suggests that also in Drosophila 'the A-rule' is valid, indicating that during DNA replication an adenine (A) is preferentially incorporated opposite to non-instructive apurinic sites.