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.

Induced mutagenic effects in the nucleotide excision repair deficient Drosophila mutant mus201(D1), expressing a truncated XPG protein.

Defects in nucleotide excision repair (NER) as defined by the UV sensitivity of xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD) patients has lead to the identification of most of the genes involved: XPA through XPG, CSA and CSB. Whereas XP patients often show an increased risk for skin cancer after exposure to sunlight, this is not the case for patients with CS and TTD. Several CS patients have been shown to carry a defect in the XPG gene. The XPG, a structure specific endonuclease makes the incision 3' of damage and is also involved in the subsequent 5'incision during the NER process. In addition, XPG plays a role in the removal of oxidative DNA damage. The Drosophila XPG gene was isolated and based on the molecular defect of a spontaneous (insertion) and an EMS induced mutant, it was shown that a mutated XPG is responsible for the Drosophila mutagen-sensitive mutants mus201. One of these mutants, mus201(D1) has been used extensively in studies of the effects and mechanisms of many chemical mutagens as well as X-rays. The results of these studies are discussed in the light of the finding that mus201p is the Drosophila homologue of XPG.

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.

Cloning and characterization of the Drosophila homolog of the xeroderma pigmentosum complementation-group B correcting gene, ERCC3.

Previously the human nucleotide excision repair gene ERCC3 was shown to be responsible for a rare combination of the autosomal recessive DNA repair disorders xeroderma pigmentosum (complementation group B) and Cockayne's syndrome (complementation group C). The human and mouse ERCC3 proteins contain several sequence motifs suggesting that it is a nucleic acid or chromatin binding helicase. To study the significance of these domains and the overall evolutionary conservation of the gene, the homolog from Drosophila melanogaster was isolated by low stringency hybridizations using two flanking probes of the human ERCC3 cDNA. The flanking probe strategy selects for long stretches of nucleotide sequence homology, and avoids isolation of small regions with fortuitous homology. In situ hybridization localized the gene onto chromosome III 67E3/4, a region devoid of known D.melanogaster mutagen sensitive mutants. Northern blot analysis showed that the gene is continuously expressed in all stages of fly development. A slight increase (2-3 times) of ERCC3Dm transcript was observed in the later stages. Two almost full length cDNAs were isolated, which have different 5' untranslated regions (UTR). The SD4 cDNA harbours only one long open reading frame (ORF) coding for ERCC3Dm. Another clone (SD2), however, has the potential to encode two proteins: a 170 amino acids polypeptide starting at the optimal first ATG has no detectable homology with any other proteins currently in the data bases, and another ORF beginning at the suboptimal second startcodon which is identical to that of SD4. Comparison of the encoded ERCC3Dm protein with the homologous proteins of mouse and man shows a strong amino acid conservation (71% identity), especially in the postulated DNA binding region and seven 'helicase' domains. The ERCC3Dm sequence is fully consistent with the presumed functions and the high conservation of these regions strengthens their functional significance. Microinjection and DNA transfection of ERCC3Dm into human xeroderma pigmentosum (c.g. B) fibroblasts and group 3 rodent mutants did not yield detectable correction. One of the possibilities to explain these negative findings is that the D.melanogaster protein may be unable to function in a mammalian repair context.

Characterization of MR (P) strains of Drosophila melanogaster: the number of intact P elements and their genetic effect.

To study the effect of mutagenic/carcinogenic agents on P-element transposition, the P strains used should be defined, especially with respect to the number of intact and functional P elements present. In this investigation, the relation between the number of complete P elements present in dysgenic males and P-insertion mutagenesis was studied in several MR (P) strains. The main conclusions from this investigation are: (1) Complete P elements can be present in the genome without genetic activity (even in a 'dysgenic' cross). As a consequence, the number of complete P elements present in particular dysgenic flies, is not necessarily an indication of their dysgenic genetic activity. (2) The MR-h12/Cy strain carries two complete P elements, one on the X chromosome without and one on the MR chromosome with genetic activity (making this strain most suitable for studies on P-transposition mechanisms).

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)

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.