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In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212–mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314–mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215ZIII-2059, that uniformly reduces the frequency of meiotic recombination to

ATM/ATR kinases act as signal transducers in eukaryotic DNA damage and replication checkpoints. Mutations in ATM/ATR homologs have pleiotropic effects that range from sterility to increased killing by genotoxins in humans, mice, and Drosophila. Here we report the generation of a null allele of mei-41, Drosophila ATM/ATR homolog, and the use of it to document a semidominant effect on a larval mitotic checkpoint and methyl methanesulfonate (MMS) sensitivity. We also tested the role of mei-41 in a recently characterized checkpoint that delays metaphase/anaphase transition after DNA damage in cellular embryos. We then compare five existing mei-41 alleles to the null with respect to known phenotypes (female sterility, cell cycle checkpoints, and MMS resistance). We find that not all phenotypes are affected equally by each allele, i.e., the functions of MEI-41 in ensuring fertility, cell cycle regulation, and resistance to genotoxins are genetically separable. We propose that MEI-41 acts not in a single rigid signal transduction pathway, but in multiple molecular contexts to carry out its many functions. Sequence analysis identified mutations, which, for most alleles, fall in the poorly characterized region outside the kinase domain; this allowed us to tentatively identify additional functional domains of MEI-41 that could be subjected to future structure-function studies of this key molecule.

We previously reported evidence for two subpopulations of several classes of steroid receptors that could be distinguished by their requirement of a low molecular weight factor (Mr=700-3000 Da) for binding to nonspecific, calf thymus DNA-cellulose [Cavanaugh, A. H. and Simons Jr., S. S., Journal of Steroid Biochemistry and Molecular Biology, 48, 433-446 (1994)]. This factor appeared to be enriched in (NH4)2SO4 precipitates of nuclear extracts. Using human progesterone receptors (PRs) and biologically active DNA sequences in a modified avidin/biotin-coupled DNA (ABCD) binding assay, we now report a factor-mediated increase in PR binding to specific DNA sites that was indistinguishable from that seen with nonspecific sites. The main advantages of this modified assay are that both kinetic and equilibrium binding of receptor-steroid complexes to DNA can be directly monitored in solution. The ability of either Sephadex G-50 chromatography or sodium arsenite to prevent that binding which is increased by added factor supported the existence of PR subpopulations that are independent of the acceptor DNA sequence. The factor was found, surprisingly, to be low concentrations (> or = 5 mM) of (NH4)2SO4, which anomalously is partially excluded from Sephadex G-10 columns, and can be mimicked by some salts but not sodium arsenite. Kinetic analyses demonstrated that the mechanism of action of salt was to accelerate the rate of binding of PR. Salt also had a much greater effect on the nonspecific binding of PR, such that the ratio of specific to nonspecific DNA binding was greatest at elevated salt concentrations (approximately 75 mM) that afforded submaximal levels of PR binding to specific DNA sites. Further analysis of the DNA-bound receptors revealed that the smaller, A-form of PR is preferentially bound to specific DNA sequences both in the presence and in the absence of various salt concentrations. Thus, the differences in DNA binding of PR +/- salt do not correlate with the preferential binding of A or B isoform. The unequal behavior of PR subpopulations and/or isoforms for binding to specific DNA sequences offers added mechanisms for selective transcriptional regulation of genes in intact cells.

International audience; The Rad50 protein is involved in the cellular response to DNA-double strand breaks (DSBs), including the detection of damage, activation of cell-cycle checkpoints, and DSB repair via recombination. It is essential for meiosis in yeast, is involved in telomere maintenance, and is essential for cellular viability in mice. Here we present the isolation, sequence and characterization of the Arabidopsis thaliana RAD50 homologue (AtRAD50) and an Arabidopsis mutant of this gene. A single copy of this gene is present in the Arabidopsis genome, located on chromosome II. Northern analysis shows a single 4.3 Kb mRNA species in all plant tissues tested, which is strongly enriched in flowers and other tissues with many dividing cells. The predicted protein presents strong conservation with the other known Rad50 homologues of the amino- and carboxy-terminal regions. Mutant plants present a sterility phenotype which co-segregates with the T-DNA insertion. Molecular analysis of the mutant plants shows that the sterility phenotype is present only in the plants homozygous for the T-DNA insertion. An in vitro mutant cell line, derived from the mutant plant, shows a clear hypersensitivity to the DNA-damaging agent methylmethane sulphonate, suggesting a role of RAD50 in double-strand break repair in plant cells. This is the first report of a plant mutated in a protein of the Rad50-Mre11-Xrs2 complex, as well as the first data suggesting the involvement of the Rad50 homologue protein in meiosis and DNA repair in plants.