J.C.J. Eeken

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.

Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination.

The RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks. Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were cloned by the polymerase chain reaction. DNA sequence analysis revealed an open reading frame of 418 amino acids for the human RAD52 homolog and of 420 amino acid residues for the mouse counterpart. The identity between the two proteins is 69% and the overall similarity 80%. The homology of the mammalian proteins with their counterparts from yeast is primarily concentrated in the N-terminal region. Low amounts of RAD52 RNA were observed in adult mouse tissues. A relatively high level of gene expression was observed in testis and thymus, suggesting that the mammalian RAD52 protein, like its homolog from yeast, plays a role in recombination. The mouse RAD52 gene is located near the tip of chromosome 6 in region G3. The human equivalent maps to region p13.3 of chromosome 12. Until now, this human chromosome has not been implicated in any of the rodent mutants with a defect in the repair of double-strand 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.

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.

Modification of MR mutator activity in repair-deficient strains of Drosophila melanogaster

In drosophila, MR (male recombination) second chromosomes are known to act as mutators and recombination-inducers in males. One explanation of MR- induced effects is that MR causes breaks at specific sites where, subsequently, insertion sequences become integrated. To examine the extent to which excision and incorporation of DNA sequences by MR is affected by enzymatic pathways involved in DNA repair, the following experiments were carried out. MR-chromosomes were introduced into males deficient for excision (mei-9a) or post-replication repair (mei-41D5) or into males carrying both repair-deficient mutations. MR activity was recorded by the occurrence of visible mutations at the sn (singed) and ras (raspberry) loci. The spontaneous mutation frequency for sn is 0.2 . 10-5 (1/490 000) and for ras, 0.4 . 10-5 (2/490 000). When MR is introduced into repair-proficient males the frequency of sn mutations is 51 . 10-5 (16/30 795) and of ras mutations is 19. 10-5 (6/30 795). In MR males deficient for both excision and post-replication repair (mei-9a mei-41D5) mutator activity is significantly enhanced; the frequency of sn mutations amounts to 225 . 10-5 (174/77 219) and of ras mutations to 135 . 10-5 (104/77 219). mei-9a or mei-41D5 alone also leads to an enhancement of the mutation frequencies, but this effect is of borderline significance.

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 effect of two chemical mutagens ENU and MMS on MR-mediated reversion of an insertion-sequence mutation in Drosophila melanogaster

The effects of two mutagens ENU and MMS characterized by different alkylation patterns have been studied on the reversion of an MR-induced singed mutation to wild-type. Reversion of this unstable singed mutation under the influence of MR is assumed to represent the removal or transposition of an insertion element. Since MR acts primarily in spermatogonia, the mutagens were fed to 1st instar larvae. Recessive lethal tests were carried out simultaneously to calibrate for the mutagenic effectiveness of the chemicals. For both powerful mutagens, it was observed that the frequency of reversion remained far below of what would have been expected on the basis of the mutagenic effectiveness, as registered in the lethal tests. Thus 1 mM ENU, 5 mM and 10 mM MMS did not affect the reversion frequency at all, and with 3 mM ENU only a doubling of the reversion frequency was observed, despite a 5-fold increase in the lethal frequency. The threshold at 1 mM EMU and the low effectiveness of 3 mM on the reversion process are taken as an indication that ENU affected the transposition process in an indirect manner, rather than the excision events themselves. The data obtained with Drosophila are consistent with the microbial observations in that mutation involving removal or transposition of an insertion element is not affected by mutagenic treatments. This finding may have consequences for the evaluation of induced genetic damage on the basis of the spontaneous load of genetic detriment in man. An incidental observation was that non-MR Cy larvae exhibited greater sensitivity to the induction of recessive lethals by MMS than MR-individuals.

Influence of the MR (mutator) factor on X-ray-induced genetic damage

The genetical effects induced by MR, in the progeny of outcrossed MR-males, include very high frequencies of visible and lethal mutations and chromosome aberrations. The hypothesis is that MR causes breaks at specific sites in the DNA where, subsequently, insertion sequences become integrated. To examine whether there exists an interaction between breaks and radiation induced lesions, MRh12/Cy males were crossed to Berlin K females and the male progeny from this cross carrying the MR or Cy chromosome were irradiated. The frequencies of X-linked recessive lethals and II-III translocations were determined. Non-irradiated MR and non-MR (Cy) male progeny were used in concurrent controls. The results show that the frequencies of II-III translocations in the MR-containing males is not significantly higher than in the controls. However, with regard to the production of recessive lethal mutations a clear synergism between MRh12 and X-irradiation was observed.

Neither enhanced removal of cyclobutane pyrimidine dimers nor strand-specific repair is found after transcription induction of the β3-tubulin gene in a Drosophila embryonic cell line Kc

Nucleotide excision repair (NER) of ultraviolet (UV) light induced cyclobutane pyrimidine dimers (CPDs) was assayed in a Drosophila melanogaster Kc subline that responds to treatment with the steroid hormone 20-hydroxyecdysone (20-OH-E; beta-ecdysone, ecdysterone). In this cell line the hormone induces transcription of the beta 3-tubulin gene which is not expressed under standard culture conditions. Cells were exposed to either 10 or 15 J/m2 UV (predominantly 254-nm) and removal of CPDs from several genes, including beta 3-tubulin, and total cellular DNA was assayed. We show that upon induction of transcription of the beta 3-tubulin gene, its repair is not enhanced. In non-treated as well as 20-OH-E treated cells, repair kinetics in beta 3-tubulin resemble those in the active genes Gart and Notch, the inactive locus white and total cellular DNA. Moreover, in the presence as well as in the absence of transcription, the separate strands of the beta 3-tubulin gene are repaired with the same rate and to the same extent: about 90% after 24 h. It can be concluded from these observations that transcription is not a prerequisite for the efficient repair of CPDs in the Drosophila embryonic Kc cell line.

The influence of deficiencies in DNA-repair on MR-mediated reversion of an insertion-sequence mutation in Drosophila melanogaster

MR is a frequently occurring mutator in Drosophila melanogaster inducing mutation by the incorporation of insertion sequences. In the presence of MR a mutation at the singed (sn) locus induced by MR, reverts to wild-type at a high frequency of 1.7%. This reversion system which presumably involves the removal of an insertion element, was used to study the effects of defective DNA repair. Thus, reversion frequencies were compared in progeny of flies with mei-9, deficient for excision repair, mei-41, deficient for post-replication repair, or with both mei-9 and mei-41. The data show that under conditions of defective DNA repair, the frequency of MR-mediated reversion, is consistently decreased in comparison to repair-proficient conditions. This effect is explained by assuming that defective repair interferes with some steps in the process of reverse mutation involving the removal of insertion sequences. The observed reduction in reversion frequency may well result from selective elimination of cells in which the reversion process has not been completed.